Heated nanowells for polynucleotide synthesis

ABSTRACT

Devices for the manufacturing of high-quality building blocks, such as oligonucleotides, are described herein. Nano-scale devices allow for selective control over reaction conditions. Further, methods and devices described herein allow for the rapid construction of large libraries of highly accurate nucleic acids.

CROSS-REFERENCE

This application claims the benefit of U.S. provisional patentapplication No. 62/575,287 filed on Oct. 20, 2017, which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 16, 2018, isnamed 44854-744_201_SL.txt and is 685 bytes in size.

BACKGROUND

De novo gene synthesis is a powerful tool for basic biological researchand biotechnology applications. While various methods are known for thedesign and synthesis of relatively short fragments in a small scale,these techniques often suffer from predictability, scalability,automation, speed, accuracy, and cost.

BRIEF SUMMARY

Provided herein are devices for polynucleotide synthesis comprising: asolid support comprising a surface; a plurality of structures forpolynucleotide extension located on the the surface, wherein eachstructure has a width of about 10 nm to about 1000 nm, wherein eachstructure is in contact with a heating unit, and wherein the heatingunit comprises at least one electrode; and a solvent distributed acrossthe surface, wherein the solvent is a polar solvent. Further providedherein are devices wherein the surface comprises at least 30,000 locifor nucleic acid synthesis. Further provided herein are devices whereinthe surface comprises at least 50,000 loci for nucleic acid synthesis.Further provided herein are devices wherein the surface comprises atleast 100,000 loci for nucleic acid synthesis. Further provided hereinare devices wherein the surface comprises at least 200,000 loci fornucleic acid synthesis. Further provided herein are devices wherein thesurface comprises at least 1,000,000 loci for nucleic acid synthesis.Further provided herein are devices wherein the one or more electrodesare addressible electrodes that heats one or more individual loci.Further provided herein are devices wherein the solid support furthercomprises a cooling unit. Further provided herein are devices whereinthe distance between the centers of any two structures is about 10 nm toabout 1000 nm. Further provided herein are devices wherein the distancebetween the centers of any two structures is about 100 nm to about 500nm. Further provided herein are devices wherein the distance between thecenters of any two structures is about 20 nm to about 300 nm. Furtherprovided herein are devices wherein the distance between the centers ofany two structures is about 100 nm. Further provided herein are deviceswherein the distance between the centers of any two structures is about200 nm. Further provided herein are devices wherein the distance betweenthe centers of any two structures is about 500 nm. Further providedherein are devices wherein each structure comprises a 3-dimensionalstructure, wherein the 3-dimensional structure is a nanowell, ananowire, a nanopost, or a nanorod. Further provided herein are deviceswherein the solvent has a density of about 0.5 to about 1.5 g/mL.Further provided herein are devices wherein the solvent comprises anitrile group. Further provided herein are devices wherein the solventis trimethylacetonitrile. Further provided herein are devices whereinthe solvent has a melting temperature of between 5 degrees C. to 18degrees C. Further provided herein are devices wherein the solvent has amelting temperature of between 10 degrees C. to 18 degrees C. Furtherprovided herein are devices wherein the solvent has a meltingtemperature of between 15 degrees C. to 18 degrees C. Further providedherein are devices wherein the device is used for polynucleotidesynthesis, wherein polynucleotide synthesis comprises a plurality ofelongation steps. Further provided herein are devices wherein thesolvent is not removed during an elongation step. Further providedherein are methods for polynucleotide synthesis using the devicesdescribed herein.

Provided herein are devices for polynucleotide synthesis comprising: asolid support comprising a surface; a plurality of structures forpolynucleotide extension located on the the surface, wherein eachstructure has a width of about 10 nm to about 1000 nm, wherein eachstructure is in contact with a heating unit, and wherein the heatingunit comprises at least one electrode; and a solvent distributed acrossthe surface, wherein the solvent has a melting temperature of no morethan about 18 degrees C. Further provided herein are devices wherein thesurface comprises at least 30,000 loci for nucleic acid synthesis.Further provided herein are devices wherein the surface comprises atleast 50,000 loci for nucleic acid synthesis. Further provided hereinare devices wherein the surface comprises at least 100,000 loci fornucleic acid synthesis. Further provided herein are devices wherein thesurface comprises at least 200,000 loci for nucleic acid synthesis.Further provided herein are devices wherein the surface comprises atleast 1,000,000 loci for nucleic acid synthesis. Further provided hereinare devices wherein the one or more electrodes are addressibleelectrodes that heats one or more individual loci. Further providedherein are devices wherein the solid support further comprises a coolingunit. Further provided herein are devices wherein the distance betweenthe centers of any two structures is about 10 nm to about 1000 nm.Further provided herein are devices wherein the distance between thecenters of any two structures is about 100 nm to about 500 nm. Furtherprovided herein are devices wherein the distance between the centers ofany two structures is about 20 nm to about 300 nm. Further providedherein are devices wherein the distance between the centers of any twostructures is about 100 nm. Further provided herein are devices whereinthe distance between the centers of any two structures is about 200 nm.Further provided herein are devices wherein the distance between thecenters of any two structures is about 500 nm. Further provided hereinare devices wherein each structure comprises a 3-dimensional structure,wherein the 3-dimensional structure is a nanowell, a nanowire, ananopost, or a nanorod. Further provided herein are devices wherein thesolvent has a density of about 0.5 to about 1.5 g/mL. Further providedherein are devices wherein the solvent is a polar solvent. Furtherprovided herein are devices wherein the solvent comprises a nitrilegroup. Further provided herein are devices wherein the solvent istrimethylacetonitrile. Further provided herein are devices wherein thesolvent has a melting temperature of between 5 degrees C. to 18 degreesC. Further provided herein are devices wherein the solvent has a meltingtemperature of between 10 degrees C. to 18 degrees C. Further providedherein are devices wherein the solvent has a melting temperature ofbetween 15 degrees C. to 18 degrees C. Further provided herein aredevices wherein the device is used for polynucleotide synthesis, whereinpolynucleotide synthesis comprises a plurality of elongation steps.Further provided herein are devices wherein the solvent is not removedduring an elongation step. Further provided herein are methods forpolynucleotide synthesis using the devices described herein.

Provided herein are devices for polynucleotide synthesis comprising: asolid support comprising a surface; a plurality of structures forpolynucleotide extension located on the the surface, wherein eachstructure has a width of about 10 nm to about 1000 nm, wherein eachstructure is in contact with a heating unit, and wherein the heatingunit comprises at least one electrode; and a solvent distributed acrossthe surface, wherein the solvent has a boiling temperature of no morethan about 82 degrees C. Further provided herein are devices wherein thesurface comprises at least 30,000 loci for nucleic acid synthesis.Further provided herein are devices wherein the surface comprises atleast 50,000 loci for nucleic acid synthesis. Further provided hereinare devices wherein the surface comprises at least 100,000 loci fornucleic acid synthesis. Further provided herein are devices wherein thesurface comprises at least 200,000 loci for nucleic acid synthesis.Further provided herein are devices wherein the surface comprises atleast 1,000,000 loci for nucleic acid synthesis. Further provided hereinare devices wherein the one or more electrodes are addressibleelectrodes that heats one or more individual loci. Further providedherein are devices wherein the solid support further comprises a coolingunit. Further provided herein are devices wherein the distance betweenthe centers of any two structures is about 10 nm to about 1000 nm.Further provided herein are devices wherein the distance between thecenters of any two structures is about 100 nm to about 500 nm. Furtherprovided herein are devices wherein the distance between the centers ofany two structures is about 20 nm to about 300 nm. Further providedherein are devices wherein the distance between the centers of any twostructures is about 100 nm. Further provided herein are devices whereinthe distance between the centers of any two structures is about 200 nm.Further provided herein are devices wherein the distance between thecenters of any two structures is about 500 nm. Further provided hereinare devices wherein each structure comprises a 3-dimensional structure,wherein the 3-dimensional structure is a nanowell, a nanowire, ananopost, or a nanorod. Further provided herein are devices wherein thesolvent has a density of about 0.5 to about 1.5 g/mL. Further providedherein are devices wherein the solvent has a boiling temperature ofbetween 0 degrees C. to 82 degrees C. Further provided herein aredevices wherein the solvent has a boiling temperature of between 30degrees C. to 82 degrees C. Further provided herein are devices whereinthe solvent has a boiling temperature of between 55 degrees C. to 82degrees C. Further provided herein are devices wherein the device isused for polynucleotide synthesis, wherein polynucleotide synthesiscomprises a plurality of elongation steps. Further provided herein aredevices wherein the solvent is not removed during an elongation step.Further provided herein are methods for polynucleotide synthesis usingthe devices described herein.

Provided herein are methods for polynucleotide synthesis, comprising:providing predetermined sequences for a library of polynucleotides;providing a substrate comprising a surface; and synthesizing the libraryof polynucleotides extending from the surface, wherein differentnucleotides are sequentially added before a deblocking step occurs.Further provided herein are methods wherein synthesizing furthercomprises a solvent, wherein the solvent undergoes at least one phasechange. Further provided herein are methods wherein the solvent is apolar solvent. Further provided herein are methods wherein the solventis trimethylacetonitrile. Further provided herein are methods whereinthe solvent has a melting temperature between 15 degrees C. to 18degrees C. Further provided herein are methods wherein the solvent has adensity of between 0.5 and 1.5 g/mL. Further provided herein are methodswherein polynucleotide synthesis further comprises a plurality ofelongation steps. Further provided herein are methods wherein thesolvent is not removed during an elongation step. Further providedherein are methods wherein at least 2 different nucleotides aresequentially added before a deblocking step occurs. Further providedherein are methods wherein at least 3 different nucleotides aresequentially added before a deblocking step occurs. Further providedherein are methods wherein at least 4 different nucleotides aresequentially added before a deblocking step occurs. Further providedherein are methods wherein all of the surface is contacted with anidentical nucleotide during an elongation step. Further provided hereinare methods wherein the at least one phase change is melting orfreezing. Further provided herein are methods wherein the at least onephase change is boiling or condensing.

Provided herein are methods for polynucleotide synthesis using thedevices described herein.

Provided herein are methods for polynucleotide synthesis, the methodcomprising: providing predetermined sequences for a library ofpolynucleotides; providing a substrate comprising a surface;synthesizing the library of polynucleotides extending from the surface,wherein a solvent in the solid phase prevents deblocking of at least onepolynucleotide extending from the at least one region of the surface,wherein the solvent has a melting temperature of no more than about 18degrees C. Further provided herein are methods wherein synthesizing thelibrary of polynucleotides extending from the surface comprisescontacting the surface with a first nucleotide phosphoramidite. Furtherprovided herein are methods wherein synthesizing the library ofpolynucleotides extending from the surface further comprises contactingthe surface with a second nucleotide phosphoramidite, wherein thesolvent is not removed between contact with the first nucleotidephosphoramidite and the second nucleotide phosphoramidite. Furtherprovided herein are methods wherein synthesizing the library ofpolynucleotides extending from the surface further comprises melting thesolvent present at the at least one region of the surface, anddeblocking at least one extended polynucleotide extending from thesurface in the at least one region. Further provided herein are methodswherein the solvent has a melting temperature of no more than about 15degrees C. Further provided herein are methods wherein the solvent has amelting temperature of no more than about 10 degrees C. Further providedherein are methods wherein the solvent has a melting temperature ofbetween 5 degrees C. to 18 degrees C. Further provided herein aremethods wherein the solvent has a melting temperature of between 10degrees C. to 18 degrees C. Further provided herein are methods whereinthe solvent has a melting temperature of between 15 degrees C. to 18degrees C. Further provided herein are methods wherein the surfacecomprises at least 30,000 loci for nucleic acid synthesis. Furtherprovided herein are methods wherein the surface comprises at least50,000 loci for nucleic acid synthesis. Further provided herein aremethods wherein the surface comprises at least 100,000 loci for nucleicacid synthesis. Further provided herein are methods wherein the surfacecomprises at least 200,000 loci for nucleic acid synthesis. Furtherprovided herein are methods wherein the surface comprises at least1,000,000 loci for nucleic acid synthesis.

Provided herein are methods for polynucleotide synthesis, the methodcomprising: providing predetermined sequences for a library ofpolynucleotides; providing a substrate comprising a surface;synthesizing the library of polynucleotides extending from the surface,wherein a solvent in the gas phase prevents deblocking of at least onepolynucleotide extending from the at least one region of the surface,wherein the solvent has a boiling temperature no more than about 82degrees C. Further provided herein are methods wherein synthesizing thelibrary of polynucleotides extending from the surface comprisescontacting the surface with a first nucleotide phosphoramidite. Furtherprovided herein are methods wherein synthesizing the library ofpolynucleotides extending from the surface further comprises contactingthe surface with a second nucleotide phosphoramidite, wherein thesolvent is not removed between contact with the first nucleotidephosphoramidite and the second nucleotide phosphoramidite. Furtherprovided herein are methods wherein synthesizing the library ofpolynucleotides extending from the surface further comprises condensingthe solvent present at the at least one region of the surface, anddeblocking at least one extended polynucleotide extending from thesurface in the at least one region. Further provided herein are methodswherein the solvent has a boiling temperature no more than about 75degrees C. Further provided herein are methods wherein the solvent has aboiling temperature no more than about 65 degrees C. Further providedherein are methods wherein the solvent has a boiling temperature ofbetween 0 degrees C. to 82 degrees C. Further provided herein aremethods wherein the solvent has a boiling temperature of between 30degrees C. to 82 degrees C. Further provided herein are methods whereinthe solvent has a boiling temperature of between 55 degrees C. to 82degrees C. Further provided herein are methods wherein the surfacecomprises at least 30,000 loci for nucleic acid synthesis. Furtherprovided herein are methods wherein the surface comprises at least50,000 loci for nucleic acid synthesis. Further provided herein aremethods wherein the surface comprises at least 100,000 loci for nucleicacid synthesis. Further provided herein are methods wherein the surfacecomprises at least 200,000 loci for nucleic acid synthesis. Furtherprovided herein are methods wherein the surface comprises at least1,000,000 loci for nucleic acid synthesis.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 depicts a rigid structure, having flat features (loci), channels,or wells, respectively.

FIG. 2 depicts a schematic for the generation of polynucleotidelibraries from cluster amplification.

FIG. 3A depicts a front cross section of a heated nanowell structure,having addressable wells.

FIG. 3B depicts a right cross section of a heated nanowell structure,having addressable wells.

FIG. 4A depicts a front cross section of a heated nanowell structure,having addressable wells.

FIG. 4B depicts a front cross section of a heated nanopost structure,having addressable nanoposts.

FIG. 5 depicts a front cross section of a heated nanorod structure,having addressable nanorods.

FIG. 6 depicts a front cross section of a nanowire structure attached toan addressable bottom contact.

FIG. 7 depicts a polynucleotide synthesis material deposition device.

FIG. 8 depicts a polynucleotide synthesis workflow.

FIG. 9 depicts a a computer system.

FIG. 10 depicts a block diagram illustrating the architecture of acomputer system.

FIG. 11 depicts a network configured to incorporate a plurality ofcomputer systems, a plurality of cell phones and personal dataassistants, and Network Attached Storage (NAS).

FIG. 12 depicts a multiprocessor computer system using a shared virtualaddress memory space.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong.

Throughout this disclosure, numerical features are presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of any embodiments. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range to the tenth of the unit of the lower limitunless the context clearly dictates otherwise. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual valueswithin that range, for example, 1.1, 2, 2.3, 5, and 5.9. This appliesregardless of the breadth of the range. The upper and lower limits ofthese intervening ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention, unless thecontext clearly dictates otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of any embodiment.As used herein, the singular forms “a,” “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises”and/or “comprising,” when used in this specification, specify thepresence of stated features, integers, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, integers, steps, operations, elements,components, and/or groups thereof. As used herein, the term “and/or”includes any and all combinations of one or more of the associatedlisted items.

Unless specifically stated or obvious from context, as used herein, theterm “about” in reference to a number or range of numbers is understoodto mean the stated number and numbers +/−10% thereof, or 10% below thelower listed limit and 10% above the higher listed limit for the valueslisted for a range.

As used herein, the terms “preselected sequence”, “predefined sequence”or “predetermined sequence” are used interchangeably. The terms meanthat the sequence of the polymer is known and chosen before synthesis orassembly of the polymer. In particular, various aspects of the inventionare described herein primarily with regard to the preparation of nucleicacids molecules, the sequence of the oligonucleotide or polynucleotidebeing known and chosen before the synthesis or assembly of the nucleicacid molecules.

Provided herein are methods and compositions for production of synthetic(i.e. de novo synthesized or chemically synthesizes) polynucleotides.The term oligonucleotide, oligo, and polynucleotide are defined to besynonymous throughout. Libraries of synthesized polynucleotidesdescribed herein may comprise a plurality of polynucleotidescollectively encoding for one or more genes or gene fragments. In someinstances, the polynucleotide library comprises coding or non-codingsequences. In some instances, the polynucleotide library encodes for aplurality of cDNA sequences. Reference gene sequences from which thecDNA sequences are based may contain introns, whereas cDNA sequencesexclude introns. Polynucleotides described herein may encode for genesor gene fragments from an organism. Exemplary organisms include, withoutlimitation, prokaryotes (e.g., bacteria) and eukaryotes (e.g., mice,rabbits, humans, and non-human primates). In some instances, thepolynucleotide library comprises one or more polynucleotides, each ofthe one or more polynucleotides encoding sequences for multiple exons.Each polynucleotide within a library described herein may encode adifferent sequence, i.e., non-identical sequence. In some instances,each polynucleotide within a library described herein comprises at leastone portion that is complementary to sequence of another polynucleotidewithin the library. Polynucleotide sequences described herein may,unless stated otherwise, comprise DNA or RNA. Provided herein aremethods and compositions for production of synthetic (i.e. de novosynthesized) genes. Libraries comprising synthetic genes may beconstructed by a variety of methods described in further detailelsewhere herein, such as PCA, non-PCA gene assembly methods orhierarchical gene assembly, combining (“stitching”) two or moredouble-stranded polynucleotides to produce larger DNA units (i.e., achassis). Libraries of large constructs may involve polynucleotides thatare at least 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60,70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500 kb long orlonger. The large constructs can be bounded by an independently selectedupper limit of about 5000, 10000, 20000 or 50000 base pairs. Thesynthesis of any number of polypeptide-segment encoding nucleotidesequences, including sequences encoding non-ribosomal peptides (NRPs),sequences encoding non-ribosomal peptide-synthetase (NRPS) modules andsynthetic variants, polypeptide segments of other modular proteins, suchas antibodies, polypeptide segments from other protein families,including non-coding DNA or RNA, such as regulatory sequences e.g.promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA,small nucleolar RNA derived from microRNA, or any functional orstructural DNA or RNA unit of interest. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, intergenic DNA, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomalRNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA(miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), whichis a DNA representation of mRNA, usually obtained by reversetranscription of messenger RNA (mRNA) or by amplification; DNA moleculesproduced synthetically or by amplification, genomic DNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probes,and primers. cDNA encoding for a gene or gene fragment referred toherein, may comprise at least one region encoding for exon sequence(s)without an intervening intron sequence found in the correspondinggenomic sequence. Alternatively, the corresponding genomic sequence to acDNA may lack an intron sequence in the first place.

Devices for Polynucleotide Synthesis

Provided herein are processes and devices for the selective synthesis ofpolynucleotides in addressable locations based on a phase changeprocess. The selectivity is achieved by blocking synthesis on an activepolynucleotide synthesis surface, by converting a reaction solventphase, such as a liquid solvent, in the reaction well to a non-reactivephase, such as a solid or gas which limits exposure to reagents in thesurrounding solvent. Heating or cooling in the reaction well providesfor a shift in the physical state of the solvent exposed to thepolynucleotide synthesis surface. During an iterative syntheticprotocol, structures such as nanostructures comprising local addressableheaters (heating units, comprising for example one or more electrodes orheating elements) are used to melt or boil the solvent, affordingaccessibility at defined locations on the synthesis surface fordownstream interactions, such as nucleoside coupling and extensionreactions.

Provided herein are structures having a surface with a plurality offeatures (loci) for polynucleotide synthesis or extension. Each featureon a structure in some instances comprises one or more smallerstructures, such as a nanostructure, for controlling the phase of thesurrounding solvent in a region of a feature. Each feature in a portionof the structure 101, may comprise a substantially planar feature 103, awell 105, or a channel. See FIG. 1. In one instance each feature of thestructure has a width of about 10 nm to about 10 μm and a distancebetween the center of each feature of about 10 nm to about 10 μm. Thecross sectional shape of the feature may comprise, without limitation, acircle, a rectangle, a triangle or a polygon. The shape of the featuremay be tapered, round, a well, a channel, a wire, a rod, a pole, a cone,or any combination thereof.

In some instances, the polynucleotides are synthesized on a cluster ofloci for polynucleotide extension, released and then subsequentlysubjected to an amplification reaction, e.g., PCR. An exemplary workflowof synthesis of polynucleotides from a cluster is depicted in FIG. 2. Asilicon plate 201 includes multiple clusters 203. Within each clusterare multiple loci 221. Polynucleotides are synthesized 207 de novo on aplate 201 from the cluster 203. Polynucleotides are cleaved 211 andremoved 213 from the plate to form a population of releasedpolynucleotides 215. The population of released polynucleotides 215 arethen amplified 217 to form a library of amplified polynucleotides 219.

In a first structure, a heated nanowell for solid-liquid phase controlprovides for polynucleotide synthesis. See FIG. 3A and FIG. 3B, device300. Each feature in a portion of the structure 301, may be asubstantially planar feature 303 (e.g., flat), a well 305, or a channel.In some instances, the substantially planar feature 303 comprises afirst material 307, a second material 309, a third material 311 and afourth material 313, wherein the third material 311 covers a firstportion of the first material 307 and the second material 309, andwherein the fourth material 313 covers a second portion of the firstmaterial 307 and the second material 309. The first 307, second 309,third 311, and fourth 313 materials comprise conductors, semiconductors,or insulators. In some instances, the first material 307 comprises asemiconducting material, such as silicon. In some instances, the secondmaterial 309 forms two or more columns of bottom addressible electrodes.In some instances, the second material 309 comprises a conductor, suchas a metal. In some instances the second material 309 comprisestungsten. In some instances, the third material 311 comprises aninsulator, such as silicon dioxide. In some instances, the fourthmaterial 313 forms two or more rows of top addressable electrodes. Insome instances, the fourth material 313 comprises a conductor, such as ametal. In some instances the third material 311 comprises titaniumnitride. Consistent with the specification, other metals,semi-conductors, and insulators are used.

In some instances, simultaneously applying a current to one or more ofthe columns of the bottom addressible electrodes 309, and one or more ofthe rows of the top addressible electrodes 313, heats one or more wells305 associated with the intersection of the corresponding column of theone or more bottom electrodes 309, and the corresponding row of the oneor more top electrodes 313. In some aspects, the first material 307 andthe bottom electrodes 309 serve to conduct heat away from a synthesissurface 302 to a cooling element (or cooling unit, or cold chuck). Insome instances, lined wells 305 act as a heater and are selectiveagainst the deposition of the nucleotide binding chemistry. In someinstances, an insulator, such as SiO₂ is used to thermally isolate thewells 305 and the top surface of the device 300. In some instances, adevice for the selective synthesis of polynucleotides comprises one ormore heating elements comprised of one or more addressible electrodes.

In a second structure, a heated nanowell for liquid-gas phase controlprovides for polynucleotide synthesis. See FIG. 4A. Each feature in aportion of the device 400, may be a well 401, or a channel. In someinstances, the well is a cylinder shape. In some instances, the device400 comprises a top contact 403, a first heating element 404, a firstmaterial 405, a second heating element 406, a bottom contact 408, and asecond material 409 wherein the top contact 403 covers a first portionof the first material 405 and a first portion of the first heatingelement 404, wherein the first heating element 404 covers a firstportion of the second heating element 406, and a second portion of thefirst material 405, wherein the first material 405 covers a firstportion of the second material 409, and wherein the bottom contact 408contacts both the second heating element 406 and the the second material409. In some instances, the top contact 403 and the bottom contact formtop 403 and bottom 408 addressable contacts, respectively. In someinstances, the top contact 403 and the bottom contact 408 comprise aconductor, such as a metal. In some instances, the first material 405and the second material 409 comprise an insulating material, such assilicon dioxide. In some instances, the first heating element 404 andthe second heating element 406 comprise semiconducting materials, suchas silicon. The semiconducting material in some instances comprises oneor more dopants, such as but not limited to phosphorus, antimony,arsenic, boron, aluminum, or indium. Alternately or in combination, thefirst heating element 404 and the second heating element 406 areconductors.

In some instances, simultaneously applying an electrical current to oneor more of the bottom addressible contacts 408, and one or more of thetop addressible contacts 403 to form an electrical path heats one ormore wells 401 associated with the intersection of the corresponding oneor more bottom contacts 408, and the corresponding one or more topcontacts 403, such as at the first heating element 404 and the secondheating element 406, respectively. In some instances, heating of asolvent 402 by applying an electrical current through the device causessolvent vaporization to form a vapor nanobubble 417 that preventssolvent contact with the synthesis surface 407, which collapses when theelectrical current flow or heating is discontinued. In some instances,the electrical path includes at least one semiconductor junction, suchas a p-n junction. In some instances, this junction determines thecurrent intensity, and improves heating element stability. In someinstances, the fourth material 406 forms a heating element, andcomprises a doped semiconductor resistor.

In a third structure, a heated nanopost for liquid-gas phase controlprovides for polynucleotide synthesis. See FIG. 4B, device 410. Eachfeature in a portion of the device 410, may be a nanopost. In someinstances, the feature 410 comprises a core contact 412, a firstmaterial 413, a surface contact 414, a heating element 415, and a secondmaterial 416 wherein the surface contact 414 covers a first portion ofthe first material 413, wherein the heating element 415 covers a firstportion of the first material 413 and a first portion of the surfacecontact 414, wherein the second material 416 covers a first portion ofthe core contact 412 and a second portion of surface contact 414. Insome instances, heating of a solvent 402 by applying an electricalcurrent through the device causes solvent vaporization to form a gasbubble 417 which prevents solvent contact with the synthesis surface407, and collapses when the electrical current flow or heating isdiscontinued. In some instances, the core contact 412, surface contact414, and the heating element 415 comprise a conductor, such as a metal.In some instances, the first material 413 and the second material 416comprise an insulating material. In some instances, the heating element415 comprises a semiconducting material.

In some instances, simultaneously applying a current to one or more topsurface contacts 414 and one or more conductive core contacts 412 heatsone or more electrical resistor sidewalls (heating elements) 415. Insome instances, heating of a solvent 402 by applying an electricalcurrent through the device causes solvent vaporization to form a vapornanobubble 417 that prevents solvent contact with the synthesis surface407 and collapses when the electrical current flow or heating isdiscontinued.

In a fourth structure, a heated nanorod for liquid-gas phase controlprovides for polynucleotide synthesis. See FIG. 5, device 500. Eachfeature in a portion of the device 500, may comprise one or morenanorods 502, including but not limited to nanowires or carbonnanotubes. The location of polynucleotide synthesis or extension 507 inFIG. 5 is shown for illustration only; synthesis or extension may takeplace anywhere on the nanorod 502. In some instances, the device 500 iscomprised of a heating element 501, a nanorod 502, a top contact 503, afirst material 504, and a bottom contact 505 wherein the top contact 503covers a first portion of first material 504, wherein the heatingelement 501 covers a second portion of the first material 504 and afirst portion of the bottom contact 505, and wherein the nanorod 502covers a third portion of the first material 504.

In some instances, the top contact 503 and the bottom contact 505 formtop 503 and bottom 505 addressable contacts, respectively. In someinstances, the top contact 503 and the bottom contact 505 comprise aconductor, such as a metal. In some instances, the first material 504comprises an insulating material. In some instances, the heating element501 comprises a semiconducting material, such as silicon. Thesemiconducting material in some instances comprises one or more dopants,such as but not limited to phosphorus, antimony, arsenic, boron,aluminum, or indium. In some instances, the heating element 501comprises a conductor, such as a metal.

In some instances, simultaneously applying a current to one or more ofthe bottom addressible contacts 505, and one or more of the topaddressible contacts 503 to form an electrical path through heatingelement 501 and solvent 506 heats the area surrounding one or morenanorods 502 or nanorod clusters. In some instances, heating a solvent506 by applying an electrical current through the device causes solventvaporization to form a vapor nanobubble 517 which collapses when theelectrical current flow or heating is discontinued. The vapor bubble 517separates the synthesis surface 507 from the solvent 506. In someinstances, the electrical path includes at least one semiconductorjunction, such as a p-n junction at the heating element 501. In someinstances, this junction determines the current intensity, and improvesheating element stability. In some instances, the heating element 501comprises a doped semiconductor resistor. In some instances, the nanorod502 provides increased surface area for nucleotide coupling, leading tohigher polynucleotide yields. In some instances, the number and scale ofnanorods 502 and electrodes may be reduced.

In a fifth structure, a nanorod on a contact provides for polynucleotidesynthesis. See FIG. 6, device 600. Each feature in a portion of thedevice 600, may be one or more nanorods 603, including but not limitedto nanowires or carbon nanotubes. In some instances, nanorods 603 are incontact with solvent 604. The location of polynucleotide synthesis orextension 607 in FIG. 6 is shown for illustration only; synthesis orextension may take place anywhere on the nanorod 603. In some instances,the substantially planar feature 600 comprises a bottom contact 601, oneor more nanorods 603, and a first material 602, wherein the firstmaterial 602 covers a first portion of the bottom contact 601. In someinstances, the bottom contact 601 forms a bottom addressable contact. Insome instances, the bottom contact 601 comprises a conductor, such as ametal. In some instances, the nanorod 603 comprises a conductor or asemiconductor. In some instances, the first material 602 comprises aninsulating material.

In some instances, the nanorods 603 comprise a conductive material. Insome instances, the nanorod feature 607 provides increased surface areafor nucleotide coupling, leading to higher polynucleotide yields. Insome instances, the number and scale of nanorods and contacts may bereduced, for example, to one nanorod with one contact. In someinstances, the bottom contact 601 is a thermal contact. In someinstances, cooling of the bottom contact 601 cools one or more nanorods607, and coats the one or more nanorods 603 with a layer of frozensolvent, that prevents solvent contact with the synthesis surface 607.

Structures for Polynucleotide Synthesis

In some instances, a well described herein has a width to depth (orheight) ratio of 20 to 0.01, wherein the width is a measurement of thewidth at the narrowest segment of the well. In some instances, a welldescribed herein has a width to depth (or height) ratio of 20 to 0.05,wherein the width is a measurement of the width at the narrowest segmentof the well. In some instances, a well described herein has a width todepth (or height) ratio of 1 to 0.01, wherein the width is a measurementof the width at the narrowest segment of the well. In some instances, awell described herein has a width to depth (or height) ratio of 0.5 to0.01, wherein the width is a measurement of the width at the narrowestsegment of the well. In some instances, a well described herein has awidth to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16,0.2, 0.5, 1, 2, 5, 10 or 20.

In some instances, a well described herein has a diameter to depth (orheight) ratio of 20 to 0.01, wherein the diameter is a measurement ofthe diameter at the narrowest segment of the well. In some instances, awell described herein has a diameter to depth (or height) ratio of 20 to0.05, wherein the diameter is a measurement of the diameter at thenarrowest segment of the well. In some instances, a well describedherein has a diameter to depth (or height) ratio of 1 to 0.01, whereinthe diameter is a measurement of the diameter at the narrowest segmentof the well. In some instances, a well described herein has a diameterto depth (or height) ratio of 0.5 to 0.01, wherein the diameter is ameasurement of the diameter at the narrowest segment of the well. Insome instances, a well described herein has a diameter to depth (orheight) ratio of about 0.01, 0.05, 0.1, 0.15, 0.16, 0.2, 0.5, 1, 2, 5,10, or 20.

In some instances, a structure described herein comprises a plurality ofwells, wherein the height or depth of the well is from about 10 nm toabout 10 μm, from about 10 nm to about 1 μm, from about 10 nm to about500 nm, from about 10 nm to about 100 nm, from about 50 nm to about 700nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm,from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, fromabout 50 nm to about 200 nm, or from about 50 nm to about 100 nm. Insome instances, the height of a well is no more than 10 μm, 5 μm, 2 μm,1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. Insome instances, the well height is about 10 nm, 20 nm, 30 nm, 40 nm, 50nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

In some instances, a structure described herein comprises a plurality ofwells, wherein the width of the well is from about 10 nm to about 10 μm,from about 10 nm to about 1 μm, from about 10 nm to about 500 nm, fromabout 10 nm to about 100 nm, from about 50 nm to about 700 nm, fromabout 50 nm to about 600 nm, from about 50 nm to about 500 nm, fromabout 50 nm to about 400 nm, from about 50 nm to about 300 nm, fromabout 50 nm to about 200 nm, or from about 50 nm to about 100 nm. Insome instances, the width of a well is no more than 10 μm, 5 μm, 2 μm, 1μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm. In someinstances, well width is about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm,70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 μm, 2 μm,5 μm, 10 μm, or more than 10 μm.

In some instances, a structure described herein comprises a plurality ofwells, wherein the diameter of the well is from about 10 nm to about 10μm, from about 10 nm to about 1 μm, from about 10 nm to about 500 nm,from about 10 nm to about 100 nm, from about 50 nm to about 700 nm, fromabout 50 nm to about 600 nm, from about 50 nm to about 500 nm, fromabout 50 nm to about 400 nm, from about 50 nm to about 300 nm, fromabout 50 nm to about 200 nm, or from about 50 nm to about 100 nm. Insome instances, the diameter of a well is no more than 10 μm, 5 μm, 2μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm.In some instances, well diameter is about 10 nm, 20 nm, 30 nm, 40 nm, 50nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

In some instances, a spot or substantially planar feature describedherein has a diameter from about 50 nm to about 1000 nm, from about 50nm to about 900 nm, from about 50 nm to about 800 nm, from about 50 nmto about 700 nm, from about 50 nm to about 600 nm, from about 50 nm toabout 500 nm, from about 50 nm to about 400 nm, from about 50 nm toabout 300 nm, from about 50 nm to about 200 nm, or from about 50 nm toabout 100 nm.

In some instances, a channel described herein has a width to depth (orheight) ratio of 20 to 0.01, wherein the channel is a measurement of thewidth at the narrowest segment of the channel. In some instances, achannel described herein has a width to depth (or height) ratio of 20 to0.05, wherein the width is a measurement of the width at the narrowestsegment of the channel. In some instances, a channel described hereinhas a width to depth (or height) ratio of 1 to 0.01, wherein the widthis a measurement of the width at the narrowest segment of the channel.In some instances, a channel described herein has a width to depth (orheight) ratio of 0.5 to 0.01, wherein the width is a measurement of thewidth at the narrowest segment of the well. In some instances, a channeldescribed herein has a width to depth (or height) ratio of about 0.01,0.05, 0.1, 0.15, 0.16, 0.2, 0.5, 1, 2, 5, 10 or 20.

In some instances, a channel described herein has a diameter to depth(or height) ratio of 20 to 0.01, wherein the diameter is a measurementof the diameter at the narrowest segment of the channel. In someinstances, a channel described herein has a diameter to depth (orheight) ratio of 20 to 0.05, wherein the diameter is a measurement ofthe diameter at the narrowest segment of the channel. In some instances,a channel described herein has a diameter to depth (or height) ratio of1 to 0.01, wherein the diameter is a measurement of the diameter at thenarrowest segment of the channel. In some instances, a channel describedherein has a diameter to depth (or height) ratio of 0.5 to 0.01, whereinthe diameter is a measurement of the diameter at the narrowest segmentof the channel. In some instances, a channel described herein has adiameter to depth (or height) ratio of about 0.01, 0.05, 0.1, 0.15,0.16, 0.2, 0.5, 1, 2, 5, 10, or 20.

In some instances, a structure described herein comprises a plurality ofchannels, wherein the height or depth of the channel is from about 10 nmto about 10 μm, from about 10 nm to about 1 μm, from about 10 nm toabout 500 nm, from about 10 nm to about 100 nm, from about 50 nm toabout 700 nm, from about 50 nm to about 600 nm, from about 50 nm toabout 500 nm, from about 50 nm to about 400 nm, from about 50 nm toabout 300 nm, from about 50 nm to about 200 nm, or from about 50 nm toabout 100 nm. In some instances, the height of a channel is no more than10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or nomore than 10 nm. In some instances, channel height is about 10 nm, 20nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300nm, 400 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

In some instances, a structure described herein comprises a plurality ofchannels, wherein the width of the channel is from about 10 nm to about10 μm, from about 10 nm to about 1 μm, from about 10 nm to about 500 nm,from about 10 nm to about 100 nm, from about 50 nm to about 700 nm, fromabout 50 nm to about 600 nm, from about 50 nm to about 500 nm, fromabout 50 nm to about 400 nm, from about 50 nm to about 300 nm, fromabout 50 nm to about 200 nm, or from about 50 nm to about 100 nm. Insome instances, the width of a channel is no more than 10 μm, 5 μm, 2μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm.In some instances, channel width is about 10 nm, 20 nm, 30 nm, 40 nm, 50nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

In some instances, a structure described herein comprises a plurality ofchannels, wherein the diameter of the channel is from about 10 nm toabout 10 μm, from about 10 nm to about 1 μm, from about 10 nm to about500 nm, from about 10 nm to about 100 nm, from about 50 nm to about 700nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm,from about 50 nm to about 400 nm, from about 50 nm to about 300 nm, fromabout 50 nm to about 200 nm, or from about 50 nm to about 100 nm. Insome instances, the diameter of a channel is no more than 10 μm, 5 μm, 2μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or no more than 10 nm.In some instances, well diameter is about 10 nm, 20 nm, 30 nm, 40 nm, 50nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm,1 μm, 2 μm, 5 μm, 10 μm, or more than 10 μm.

In some instances, the width of a feature (e.g., substantially planarfeature, well, channel, or other feature supporting polynucleotidesynthesis) is from about 10 nm to about 10 μm, from about 100 nm toabout 10 μm, from about 200 nm to about 1 μm, from about 50 nm to about500 nm, from about 50 nm to about 200 μm, or from about 10 nm to about100 nm, for example, about 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100nm, 50 nm, 20 nm, or 10 nm. In some instances, the width of a feature isno more than about 10 μm, 5 μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50nm or 10 nm. In some instances, the distance between the center of twoadjacent features is from about 10 nm to about 10 μm, 20 nm to about 5μm, from about 50 nm to about 2 nm, from about 100 nm to about 1 μm,from about 200 nm to about 500 nm, from about 200 nm to about 1 μm, fromabout 200 nm to about 750 nm, or from about 300 nm to about 600 nm, forexample, about 500 nm. In some instances, the total width of a featureis about 10 nm, 20 nm, 50 nm, 100 nm, 200 nm, 500 nm, 1 μm, 5 μm, 10 μm,20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. Insome instances, the total width of a feature is about 10 nm to 1 μm, 20nm to 500 nm, or 50 nm to 100 nm.

In some instances, the width of a structure (e.g., substantially planarstructure, well, channel, nanowell, nanorod, nanopost, or othernanostructure supporting polynucleotide synthesis) is from about 10 nmto about 10 μm, from about 100 nm to about 10 μm, from about 200 nm toabout 1 μm, from about 50 nm to about 500 nm, from about 50 nm to about200 μm, or from about 10 nm to about 100 nm, for example, about 10 μm, 5μm, 2 μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm, 20 nm, or 10 nm. In someinstances, the width of a structure is no more than about 10 μm, 5 μm, 2μm, 1 μm, 500 nm, 200 nm, 100 nm, 50 nm or 10 nm. In some instances, thedistance between the center of two adjacent structures is from about 10nm to about 10 μm, 20 nm to about 5 μm, from about 50 nm to about 2 nm,from about 100 nm to about 1 μm, from about 200 nm to about 500 nm, fromabout 200 nm to about 1 μm, from about 200 nm to about 750 nm, or fromabout 300 nm to about 600 nm, for example, about 500 nm. In someinstances, the total width of a structure is about 10 nm, 20 nm, 50 nm,100 nm, 200 nm, 500 nm, 1 μm, 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm,60 μm, 70 μm, 80 μm, 90 μm, or 100 μm. In some instances, the totalwidth of a structure is about 10 nm to 1 μm, 20 nm to 500 nm, or 50 nmto 100 nm.

Surfaces for Polynucleotide Synthesis

In some instances, each feature supports the synthesis of a populationof polynucleotides having a different sequence than a population ofpolynucleotides grown on another feature. Provided herein are surfaceswhich comprise at least 10, 100, 256, 500, 1,000, 2,000, 3,000, 4,000,5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000,14,000, 15,000, 20,000, 30,000, 40,000, 50,000 or more clusters.Provided herein are surfaces which comprise more than 2,000; 5,000;10,000; 20,000; 30,000; 50,000; 100,000; 200,000; 300,000; 400,000;500,000; 600,000; 700,000; 800,000; 900,000; 1,000,000; 5,000,000; or10,000,000 or more distinct features. In some instances, each clusterincludes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,100, 120, 130, 150, 200, 500 or more features. In some instances, eachcluster includes 50 to 500, 50 to 200, 50 to 150, or 100 to 150features. In some instances, each cluster includes 100 to 150 features.In exemplary arrangements, each cluster includes 109, 121, 130 or 137features. In some instances, each structure within a feature (such as ananostructure) supports the synthesis of a population of polynucleotideshaving a different sequence than a population of polynucleotides grownon another structure, within the same feature. Provided herein arefeatures which in some instances each comprise at least 1; 2; 5; 10; 20;50; 100; 200, 500, 1,000, 2,000, 5,000, 10,000, 20,000 or more than200,000 distinct nanostructures. In some instances, each featurecomprises about 10 to about 500, about 50 to about 250, about 10 toabout 1000, or about 1 to about 50 nanostructures.

Provided herein are features having a width at the longest segment of 10nm to 1 μm. In some instances, the features have a width at the longestsegment of about 10, 20, 30, 35, 40, 45, 50, 55 or 60 nm. In someinstances, the features are channels having multiple segments, whereineach segment has a center to center distance apart of 5 to 50 nm. Insome instances, the center to center distance apart for each segment isabout 5, 10, 15, 20 or 25 nm.

In some instances, the number of distinct polynucleotides synthesized onthe surface of a structure described herein is dependent on the numberof distinct features available in the substrate. In some instances, thedensity of features within a cluster of a substrate is at least or about1 feature per mm², 10 features per mm², 25 features per mm², 50 featuresper mm², 65 features per mm², 75 features per mm², 100 features per mm²,130 features per mm², 150 features per mm², 175 features per mm², 200features per mm², 300 features per mm², 400 features per mm², 500features per mm², 1,000 features per mm², 2,000 features per mm², 5,000features per mm², 10,000 features per mm², 100,000 features per mm²,1,000,000 features per mm² or more than 1,000,000 features per mm². Insome instances, a substrate comprises from about 10 features per mm² toabout 500 features per mm², from about 25 features per mm² to about 400features per mm², from about 50 features per mm² to about 500 featuresper mm², from about 100 features per mm² to about 500 features per mm²,from about 150 features per mm² to about 500 features per mm², fromabout 10 features per mm² to about 250 features per mm², from about 50features per mm² to about 250 features per mm², from about 10 featuresper mm² to about 200 features per mm², or from about 50 features per mm²to about 200 features per mm². In some instances, the density offeatures within a cluster of a substrate is at least or about 1 featureper μm², 10 features per μm², 25 features per μm², 50 features per μm²,65 features per μm², 75 features per μm², 100 features per μm², 130features per μm², 150 features per μm², 175 features per μm², 200features per μm², 300 features per μm², 400 features per μm², 500features per μm², 1,000 features per μm², 2,000 features per μm², 5,000features per μm², 10,000 features per μm², 100,000 features per μm²,1,000,000 features per μm² or more than 1,000,000 features per μm². Insome instances, a substrate comprises from about 10 features per μm² toabout 500 features per μm², from about 25 features per μm² to about 400features per μm², from about 50 features per μm² to about 500 featuresper μm², from about 100 features per μm² to about 500 features per μm²,from about 150 features per μm² to about 500 features per μm², fromabout 10 features per μm² to about 250 features per μm², from about 50features per μm² to about 250 features per μm², from about 10 featuresper μm² to about 200 features per μm², or from about 50 features per μm²to about 200 features per μm². In some instances, the distance betweenthe centers of two adjacent features within a cluster is from about 10μm to about 500 μm, from about 10 μm to about 200 μm, or from about 10μm to about 100 μm. In some instances, the distance between two centersof adjacent features is greater than about 10 μm, 20 μm, 30 μm, 40 μm,50 μm, 60 μm, 70 μm, 80 μm, 90 μm or 100 μm. In some instances, thedistance between the centers of two adjacent features is less than about200 μm, 150 μm, 100 μm, 80 μm, 70 μm, 60 μm, 50 μm, 40 μm, 30 μm, 20 μmor 10 μm. In some instances, the distance between the centers of twoadjacent features within a cluster is from about 10 nm to about 1000 nm,from about 10 nm to about 500 nm, 10 nm to about 200 nm, or from about10 nm to about 100 nm. In some instances, the distance between twocenters of adjacent features is greater than about 10 nm, 20 nm, 30 nm,40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm or 100 nm. In some instances,the distance between the centers of two adjacent features is less thanabout 500 nm, 200 nm, 150 nm, 100 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm,30 nm, 20 nm or 10 nm. In some instances, each square meter of astructure described herein allows for at least about 10⁷, 10⁸, 10⁹,10¹⁰, 10¹¹, or at least about 10¹² features, where each feature supportsone polynucleotide. In some instances, each square meter of a structuredescribed herein allows for at least about 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, orat least about 10¹² features, where each feature supports a plurality ofdifferent polynucleotides. In some instances, 10⁹ polynucleotides aresupported on less than about 6, 5, 4, 3, 2 or 1 m² of a structuredescribed herein.

In some instances, a structure described herein provides support for thesynthesis of more than 2,000; 5,000; 10,000; 20,000; 30,000; 50,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000;2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000;5,000,000; 10,000,000; 100,000,000 or more non-identicalpolynucleotides. In some instances, the structure provides support forthe synthesis of more than 2,000; 5,000; 10,000; 20,000; 50,000;100,000; 200,000; 300,000; 400,000; 500,000; 600,000; 700,000; 800,000;900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000;2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000;5,000,000; 10,000,000; 100,000,000 or more polynucleotides encoding fordistinct sequences. In some instances, at least a portion of thepolynucleotides have an identical sequence or are configured to besynthesized with an identical sequence. In some instances, the structureprovides a surface environment for the growth of polynucleotides havingat least about 50, 60, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450,475, 500, 1,000, 2,000 bases or more than 2,000 bases. In someinstances, the structure provides a surface environment for the growthof polynucleotides having between 50 and 2,000, bases, 50 and 1,000, 50and 500, 50 and 250, or between 100 and 1,000, 100 and 500, or between100 and 300 bases.

In some instances, polynucleotides are synthesized on distinct featuresof a structure, wherein each feature supports the synthesis of apopulation of polynucleotides. In some instances, each feature supportsthe synthesis of a population of polynucleotides having a differentsequence than a population of polynucleotides grown on another locus. Insome instances, the features of a structure are located within aplurality of clusters. In some instances, a structure comprises at least10, 500, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000,10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 20,000, 30,000, 40,000,50,000 or more clusters. In some instances, a structure comprises morethan 2,000; 5,000; 10,000; 100,000; 200,000; 300,000; 400,000; 500,000;600,000; 700,000; 800,000; 900,000; 1,000,000; 1,100,000; 1,200,000;1,300,000; 1,400,000; 1,500,000; 1,600,000; 1,700,000; 1,800,000;1,900,000; 2,000,000; 300,000; 400,000; 500,000; 600,000; 700,000;800,000; 900,000; 1,000,000; 1,200,000; 1,400,000; 1,600,000; 1,800,000;2,000,000; 2,500,000; 3,000,000; 3,500,000; 4,000,000; 4,500,000;5,000,000; or 10,000,000 or more distinct features. In some instances,each cluster includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 120, 130, 150 or more features (loci). In someinstances, each cluster includes 50 to 500, 100 to 150, or 100 to 200features. In some instances, each cluster includes 109, 121, 130 or 137features. In some instances, each cluster includes 5, 6, 7, 8, 9, 10, 11or 12 features. In some instances, polynucleotides from distinctfeatures within one cluster have sequences that, when assembled, encodefor a contiguous longer polynucleotide of a predetermined sequence.

Structure Size

In some instances, a structure described herein is about the size of astandard 96 well plate, for example between about 100 and 200 mm bybetween about 50 and 150 mm. In some instances, a structure describedherein has a diameter less than or equal to about 1000 mm, 500 mm, 450mm, 400 mm, 300 mm, 250 nm, 200 mm, 150 mm, 100 mm or 50 mm. In someinstances, the diameter of a substrate is between about 25 mm and 1000mm, between about 25 mm and about 800 mm, between about 25 mm and about600 mm, between about 25 mm and about 500 mm, between about 25 mm andabout 400 mm, between about 25 mm and about 300 mm, or between about 25mm and about 200. Non-limiting examples of substrate size include about300 mm, 200 mm, 150 mm, 130 mm, 100 mm, 76 mm, 51 mm and 25 mm. In someinstances, a substrate has a planar surface area of at least about 100mm²; 200 mm²; 500 mm²; 1,000 mm²; 2,000 mm²; 5,000 mm²; 10,000 mm²;12,000 mm²; 15,000 mm²; 20,000 mm²; 30,000 mm²; 40,000 mm²; 50,000 mm²or more. In some instances, a substrate has a thickness between about 50mm and about 2000 mm, between about 50 mm and about 1000 mm, betweenabout 100 mm and about 1000 mm, between about 200 mm and about 1000 mm,or between about 250 mm and about 1000 mm. Non-limiting examples ofthickness include 275 mm, 375 mm, 525 mm, 625 mm, 675 mm, 725 mm, 775 mmand 925 mm. In some instances, the thickness of the substrate varieswith diameter and depends on the composition of the substrate. Forexample, a structure comprising materials other than silicon may have adifferent thickness than a silicon structure of the same diameter.Structure thickness may be determined by the mechanical strength of thematerial used and the structure must be thick enough to support its ownweight without cracking during handling. In some instances, a structureis more than about 1, 2, 3, 4, 5, 10, 15, 30, 40, 50 feet in any onedimension.

In some instances, a structure described herein comprises ananostructure, for example between about 10 and 200 nm by between about10 and 150 nm. In some instances, a structure described herein has adiameter less than or equal to about 1000 nm, 500 nm, 450 nm, 400 nm,300 nm, 250 nm, 200 nm, 150 nm, 100 nm or 50 nm. In some instances, thediameter of a structure is between about 10 nm and 1000 nm, betweenabout 10 nm and about 800 nm, between about 10 nm and about 600 nm,between about 10 nm and about 500 nm, between about 10 nm and about 400nm, between about 10 nm and about 300 nm, or between about 10 mm andabout 200 nm. Non-limiting examples of structure size include about 300nm, 200 nm, 150 nm, 130 nm, 100 nm, 76 nm, 51 nm 25 nm, and 10 nm. Insome instances, a structure has a planar surface area of at least about100 nm²; 200 nm²; 500 nm²; 1,000 nm²; 2,000 nm²; 5,000 nm²; 10,000 nm²;12,000 nm²; 15,000 nm²; 20,000 nm²; 30,000 nm²; 40,000 nm²; 50,000 nm²or more. In some instances, a structure has a thickness between about 10nm and about 2000 nm, between about 50 nm and about 1000 mm, betweenabout 100 nm and about 1000 nm, between about 200 nm and about 1000 nm,or between about 250 nm and about 1000 nm. Non-limiting examples ofthickness include 50 nm, 100 nm, 275 nm, 375 nm 525 nm, 625 nm, 675 nm,725 nm, 775 nm and 925 nm.

Materials

Provided herein are devices comprising a surface, wherein the surface ismodified to support polynucleotide synthesis at predetermined locationsand with a resulting low error rate, a low dropout rate, a high yield,and a high oligo representation. In some instances, surfaces of a devicefor polynucleotide synthesis provided herein are fabricated from avariety of materials capable of modification to support a de novopolynucleotide synthesis reaction. In some instances, the devices aresufficiently conductive, e.g., are able to form uniform electric fieldsacross all or a portion of the device. A device described herein maycomprise a flexible material. Exemplary flexible materials include,without limitation, modified nylon, unmodified nylon, nitrocellulose,and polypropylene. A device described herein may comprise a rigidmaterial. Exemplary rigid materials include, without limitation, glass,fuse silica, silicon, silicon dioxide, silicon nitride, metal nitride,metal silicide, metal carbide, metal oxide, plastics (for example,polytetrafluoroethylene, polypropylene, polystyrene, polycarbonate, andblends thereof), and metals (for example, gold, platinum). In someinstances, metal oxides include TiO₂, Ta₂O₅, Nb₂O₅, Al₂O₃, BaO, Y₂O₃,HfO₂, SrO or other metal oxide known in the art. In some instances,metal carbides include TiC, WC, ThC₂, ThC, VC, W₂C, ZrC, HfC, NbC, TaC,Ta₂C, or other metal carbide known in the art. In some instances, metalnitrides include GaN, InN, BN, Be₃N₂, Cr₂N, MoN, Si₃N₄, TaN, Th₂N₂, VN,ZrN, TiN, HfN, NbC, WN, TaN, or other metal nitride known in the art.Devices disclosed herein are in some instances fabricated from amaterial comprising silicon, polystyrene, agarose, dextran, cellulosicpolymers, polyacrylamides, polydimethylsiloxane (PDMS), glass, or anycombination thereof. In some instances, a device disclosed herein ismanufactured with a combination of materials listed herein or any othersuitable material known in the art.

A listing of tensile strengths for exemplary materials described hereinis provides as follows: nylon (70 MPa), nitrocellulose (1.5 MPa),polypropylene (40 MPa), silicon (268 MPa), polystyrene (40 MPa), agarose(1-10 MPa), polyacrylamide (1-10 MPa), polydimethylsiloxane (PDMS)(3.9-10.8 MPa). Solid supports described herein can have a tensilestrength from 1 to 300, 1 to 40, 1 to 10, 1 to 5, or 3 to 11 MPa. Solidsupports described herein can have a tensile strength of about 1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 20, 25, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 270, or more MPa. In some instances, a device describedherein comprises a solid support for polynucleotide synthesis that is inthe form of a flexible material capable of being stored in a continuousloop or reel, such as a tape or flexible sheet.

Young's modulus measures the resistance of a material to elastic(recoverable) deformation under load. A listing of Young's modulus forstiffness of exemplary materials described herein is provides asfollows: nylon (3 GPa), nitrocellulose (1.5 GPa), polypropylene (2 GPa),silicon (150 GPa), polystyrene (3 GPa), agarose (1-10 GPa),polyacrylamide (1-10 GPa), polydimethylsiloxane (PDMS) (1-10 GPa). Solidsupports described herein can have a Young's moduli from 1 to 500, 1 to40, 1 to 10, 1 to 5, or 3 to 11 GPa. Solid supports described herein canhave a Young's moduli of about 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,20, 25, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 400, 500 GPa, ormore. As the relationship between flexibility and stiffness are inverseto each other, a flexible material has a low Young's modulus and changesits shape considerably under load.

In some instances, a device disclosed herein comprises a silicon dioxidebase and a surface layer of silicon oxide. Alternatively, the device mayhave a base of silicon oxide. Surface of the device provided here may betextured, resulting in an increase overall surface area forpolynucleotide synthesis. Devices described herein may comprise at least5%, 10%, 25%, 50%, 80%, 90%, 95%, or 99% silicon. A device disclosedherein may be fabricated from a silicon on insulator (SOI) wafer.

Provided herein are devices for polynucleotide synthesis comprising astructure fabricated from any one or more of a variety of materials. Incertain instances, the materials from which the substrates/solidsupports comprise are fabricated to exhibit a low level ofpolynucleotide binding. In some situations, materials that aretransparent to visible and/or UV light can be employed. Materials thatare sufficiently conductive (conductors), e.g. those that can formuniform electric fields across all or a portion of the substrates/solidssupport described herein, can be utilized. In some instances, suchmaterials may be connected to an electric ground. In some instances, thesubstrate or solid support can be heat conductive or insulated. Thematerials can be chemical resistant and heat resistant to supportchemical or biochemical reactions such as a series of polynucleotidesynthesis reactions.

In some instances, conductive or semiconductive materials(semiconductors) include but are not limited to one or more of titaniumsilicon nitride, titanium nitride, tungsten nitride, tantulum nitride,tantulum silicon nitride, titanium, platinum silicide, or otherconductive materials. In instances materials include but are not limitedto one or more of aluminumcarbides, carbides, nitrides, oxides,silicides, siliconitrides, phosphides, or other non-metal or metalloidsused as components of conductive materials. In some instances, exemplarymaterials comprise (non-limiting) one or more of the elements oftungsten, cobalt, iridium, molybdenum, nickel, platinum, rhenium,ruthenium, tantulum, titanium, or other, metals used as components ofconductive materials. In some instances, materials comprise mixtures ofmetals, non-metals, or metalloids. In some instances, dopants are addedto the semiconductive material. Dopants include but are not limited tophosphorus, antimony, arsenic, boron, aluminum, indium, or other elementconsistent with the specification. For nanostructures such as nanorods,nanowires, or nanotubes, materials of interest include conductors,semiconductors, or insulators. This includes without limitation metallicelements (for example, nickel, copper, silver, gold, platinum),semiconducting materials (for example, silicon, zinc oxide, germanium,gallium phosphide, indium nitride), or insulating materials (forexample, silicon dioxide, or titanium dioxide). Conductors,semiconductors, or insulators may be manufactured with a combination ofmaterials listed herein or any other suitable material known in the art.

For rigid materials, specific materials of interest include: glass;fused silica; silicon, plastics (for example polytetraflouroethylene,polypropylene, polystyrene, polycarbonate, and blends thereof, and thelike); metals (for example, gold, platinum, and the like). The structurecan be fabricated from a material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers,polyacrylamides, polydimethylsiloxane (PDMS), and glass. Thesubstrates/solid supports, microstructures, reactors, or otherpolynucleotide synthesis structure therein may be manufactured with acombination of materials listed herein or any other suitable materialknown in the art.

Exemplary flexible materials for structures described herein include,without limitation, nylon (unmodified nylon, modified nylon, clearnylon), nitrocellulose, polypropylene, polycarbonate, polyethylene,polyurethane, polystyrene, acetal, acrylic, acrylonitrile, butadienestyrene (ABS), polyester films such as polyethylene terephthalate,polymethyl methacrylate or other acrylics, polyvinyl chloride or othervinyl resin, transparent PVC foil, transparent foil for printers,Poly(methyl methacrylate) (PMMA), methacrylate copolymers, styrenicpolymers, high refractive index polymers, fluorine-containing polymers,polyethersulfone, polyimides containing an alicyclic structure, rubber,fabric, metal foils, and any combination thereof. Various plasticizersand modifiers may be used with polymeric substrate materials to achieveselected flexibility characteristics.

Flexible structures described herein may comprise a plastic material. Insome instances, the structure comprises a thermoplastic material.Non-limiting examples of thermoplastic materials include acrylic,acrylonitrile butadiene styrene, nylon, polylactic acid,polybenzimidazole, polycarbonate, polyether sulfone, polyetheretherketone, polyetherimide, polyethylene, polyphenylene oxide, polyphenylenesulfide, polypropylene, polystyrene, polyvinyl chloride, andpolytetrafluoroethylene. In some instances, the substrate comprises athermoplastic material in the polyaryletherketone (PEAK) family.Non-limiting examples of PEAK thermoplastics include polyetherketone(PEK), polyetherketoneketone (PEKK), poly(ether ether ketone ketone)(PEEKK), polyether ether ketone (PEEK), andpolyetherketoneetherketoneketone (PEKEKK). In some instances, thestructure comprises a thermoplastic material compatible with toluene. Insome instances, the flexibility of the plastic material is increased bythe addition of a plasticizer. An example of a plasticizer is anester-based plasticizer, such as phthalate. Phthalate plasticizersinclude bis(2-ethylhexyl) phthalate (DEHP), diisononly phthalate (DINP),di-n-butyl phthalate (DnBP, DBP), butyl benzyl phthalate (BBzP),diisodecyl phthalate (DIDP), dioctyl phthalate (DOP, DnOP), diisooctylphthalate (DIOP), diethyl phthalate (DEP), diisobutyl phthalate (DIBP),and di-n-hexyl phthalate. In some instances, modification of thethermoplastic polymer through copolymerization or through the additionof non-reactive side chains to monomers before polymerization alsoincreases flexibility.

Provided herein are flexible structures which may further comprise afluoroelastomer. Materials having about 80% fluoroelastomers aredesignated as FKMs. Fluoroelastomers include perfluoro-elastomers(FFKMs) and tetrafluoroethylene/propylene rubbers (FEPM).

Fluoroelastomers have five known types. Type 1 FKMs are composed ofvinylidene fluoride (VDF) and hexafluoropropylene (HFP) and theirfluorine content typically is around 66% by weight. Type 2 FKMs arecomposed of VDF, HFP, and tetrafluoroethylene (TFE) and typically havebetween about 68% and 69% fluorine. Type 3 FKMs are composed of VDF,TFE, and perfluoromethylvinylether (PMVE) and typically have betweenabout 62% and 68% fluorine. Type 4 FKMs are composed of propylene, TFE,and VDF and typically have about 67% fluorine. Type 5 FKMs are composedof VDF, HFP, TFE, PMVE, and ethylene.

In some instances, a substrate disclosed herein comprises a computerreadable material. Computer readable materials include, withoutlimitation, magnetic media, reel-to-reel tape, cartridge tape, cassettetape, flexible disk, paper media, film, microfiche, continuous tape(e.g., a belt) and any media suitable for storing electronicinstructions. In some instances, the substrate comprises magneticreel-to-reel tape or a magnetic belt. In some instances, the substratecomprises a flexible printed circuit board.

Structures described herein may be transparent to visible and/or UVlight. In some instances, structures described herein are sufficientlyconductive to form uniform electric fields across all or a portion of astructure. In some instances, structures described herein are heatconductive or insulated. In some instances, the structures are chemicalresistant and heat resistant to support a chemical reaction such as apolynucleotide synthesis reaction. In some instances, the substrate ismagnetic. In some instances, the structures comprise a metal or a metalalloy.

Structures for polynucleotide synthesis may be over 1, 2, 5, 10, 30, 50or more feet long in any dimension. In the case of a flexible structure,the flexible structure is optionally stored in a wound state, e.g., in areel. In the case of a large structure, e.g., greater than 1 foot inlength, the structure can be stored vertically or horizontally.

Material Deposition Systems

Provided herein are systems and devices for the deposition and storageof biomolecules on a structure described herein. In some instances, thebiomolecules are polynucleotides that store encoded information in theirsequences. In some instances, the system comprises a surface of astructure to support biomolecule attachment and/or a device forapplication of a biomolecule to the surface of the substrate. In anexample, the device for biomolecule application is a polynucleotidesynthesizer. In some instances, the system comprises a device fortreating the substrate with a fluid, for example, a flow cell. In someinstances, the system comprises a device for moving the substratebetween the application device and the treatment device. For instanceswhere the substrate is a reel-to-reel tape, the system may comprise twoor more reels that allow for access of different portions of thesubstrate to the application and optional treatment device at differenttimes.

A first example of a polynucleotide material deposition system forpolynucleotide synthesis is shown in FIG. 7. The system includes amaterial deposition device that moves in the X-Y direction to align withthe location of the substrate. The material deposition device can alsomove in the Z direction to seal with the substrate, forming a resolvedreactor. A resolved reactor is configured to allow for the transfer offluid, including polynucleotides and/or reagents, from the substrate toa capping element and/or vice versa. As shown in FIG. 7, fluid may passthrough either or both the substrate and the capping element andincludes, without limitation, coupling reagents, capping reagents,oxidizers, de-blocking agents, acetonitrile and nitrogen gas. Examplesof devices that are capable of high resolution droplet depositioninclude the printhead of inkjet printers and laser printers. The devicesuseful in the systems and methods described herein achieve a resolutionfrom about 100 dots per inch (DPI) to about 50,000 DPI; from about 100DPI to about 20,000 DPI; from about 100 DPI to about 10,000 DPI; fromabout 100 DPI to about 5,000 DPI; from about 1,000 DPI to about 20,000DPI; or from about 1,000 DPI to about 10,000 DPI. In some instances, thedevices have a resolution at least about 1,000; 2,000; 3,000; 4,000;5,000; 10,000; 12,000 DPI, or 20,000 DPI. The high resolution depositionperformed by the device is related to the number and density of eachnozzle that corresponds to a feature of the substrate.

An exemplary process workflow for de novo synthesis of a polynucleotideon a substrate using a polynucleotide synthesizer is shown in FIG. 8.Droplets comprising polynucleotide synthesis reagents are released fromthe material deposition device to the substrate in a stepwise manner,wherein the material deposition device has a piezo ceramic material andelectrodes to convert electrical signals into a mechanical signal forreleasing the droplets. The droplets are released to specific locationson the surface of the substrate one nucleobase at a time to generate aplurality of synthesized polynucleotides having predetermined sequencesthat encode data. In some instances, the synthesized polynucleotides arestored on the substrate. Nucleic acid reagents may be deposited on thesubstrate surface in a non-continuous, or drop-on-demand method.Examples of such methods include the electromechanical transfer method,electric thermal transfer method, and electrostatic attraction method.In the electromechanical transfer method, piezoelectric elementsdeformed by electrical pulses cause the droplets to be ejected. In theelectric thermal transfer method, bubbles are generated in a chamber ofthe device, and the expansive force of the bubbles causes the dropletsto be ejected. In the electrostatic attraction method, electrostaticforce of attraction is used to eject the droplets onto the substrate. Insome instances, the drop frequency is from about 5 KHz to about 500 KHz;from about 5 KHz to about 100 KHz; from about 10 KHz to about 500 KHz;from about 10 KHz to about 100 KHz; or from about 50 KHz to about 500KHz. In some instances, the frequency is less than about 500 KHz, 200KHz, 100 KHz, or 50 KHz.

The size of the droplets dispensed correlates to the resolution of thedevice. In some instances, the devices deposit droplets of reagents atsizes from about 0.01 pl to about 20 pl, from about 0.01 pl to about 10pl, from about 0.01 pl to about 1 pl, from about 0.01 pl to about 0.5pl, from about 0.01 pl to about 0.01 pl, or from about 0.05 pl to about1 pl. In some instances, the droplet size is less than about 1 pl, 0.5pl, 0.2 pl, 0.1 pl, or 0.05 pl. The size of droplets dispensed by thedevice is correlated to the diameters of deposition nozzles, whereineach nozzle is capable of depositing a reagent onto a feature of thesubstrate. In some instances, a deposition device of a polynucleotidesynthesizer comprises from about 100 to about 10,000 nozzles; from about100 to about 5,000 nozzles; from about 100 to about 3,000 nozzles; fromabout 500 to about 10,000 nozzles; or from about 100 to about 5,000nozzles. In some instances, the deposition device comprises greater than1,000; 2,000; 3,000; 4,000; 5,000; or 10,000 nozzles. In some instances,each material deposition device comprises a plurality of nozzles, whereeach nozzle is optionally configured to correspond to a feature on asubstrate. Each nozzle may deposit a reagent component that is differentfrom another nozzle. In some instances, each nozzle deposits a dropletthat covers one or more features of the substrate. In some instances,one or more nozzles are angled. In some instances, multiple depositiondevices are stacked side by side to achieve a fold increase inthroughput. In some instances, the gain is 2×, 4×, 8× or more. Anexample of a deposition device is Samba Printhead (Fujifilm). A SambaPrinthead may be used with the Samba Web Administration Tool (SWAT).

The number of deposition sites may be increased by using and rotatingthe same deposition device by a certain degree or saber angle. Byrotating the deposition device, each nozzle is jetted with a certainamount of delay time corresponding to the saber angle. Thisunsynchronized jetting creates a cross talk among the nozzles.Therefore, when the droplets are jetting at a certain saber angledifferent from 0 degrees, the droplet volume from the nozzle could bedifferent.

In some arrangements, the configuration of a polynucleotide synthesissystem allows for a continuous polynucleotide synthesis process thatexploits the flexibility of a substrate for traveling in a reel-to-reeltype process. This synthesis process operates in a continuous productionline manner with the substrate travelling through various stages ofpolynucleotide synthesis using one or more reels to rotate the positionof the substrate. In an exemplary embodiment, a polynucleotide synthesisreaction comprises rolling a substrate: through a solvent bath, beneatha deposition device for phosphoramidite deposition, through a bath ofoxidizing agent, through an acetonitrile wash bath, and through adeblock bath. Optionally, the tape is also traversed through a cappingbath. A reel-to-reel type process allows for the finished product of asubstrate comprising synthesized polynucleotides to be easily gatheredon a take-up reel, where it can be transported for further processing orstorage.

In some arrangements, polynucleotide synthesis proceeds in a continuousprocess as a continuous flexible tape is conveyed along a conveyor beltsystem. Similar to the reel-to-reel type process, polynucleotidesynthesis on a continuous tape operates in a production line manner,with the substrate travelling through various stages of polynucleotidesynthesis during conveyance. However, in a conveyor belt process, thecontinuous tape revisits a polynucleotide synthesis step without rollingand unrolling of the tape, as in a reel-to-reel process. In somearrangements, polynucleotide synthesis steps are partitioned into zonesand a continuous tape is conveyed through each zone one or more times ina cycle. For example, a polynucleotide synthesis reaction may comprise(1) conveying a substrate through a solvent bath, beneath a depositiondevice for phosphoramidite deposition, through a bath of oxidizingagent, through an acetonitrile wash bath, and through a block bath in acycle; and then (2) repeating the cycles to achieve synthesizedpolynucleotides of a predetermined length. After polynucleotidesynthesis, the flexible substrate is removed from the conveyor beltsystem and, optionally, rolled for storage. Rolling may be around areel, for storage.

In an exemplary arrangement, a flexible substrate comprisingthermoplastic material is coated with nucleoside coupling reagent. Thecoating is patterned into features such that each feature has diameterof about 10 μm, with a center-to-center distance between two adjacentfeatures of about 21 μm. In this instance, the feature size issufficient to accommodate a sessile drop volume of 0.2 pl during apolynucleotide synthesis deposition step. In some instances, the featuredensity is about 2.2 billion features per m² (1 feature/441×10⁻¹² m²).In some instances, a 4.5 m² substrate comprise about 10 billionfeatures, each with a 10 μm diameter.

In another exemplary arrangement, a substrate comprising nanostructuresis coated with nucleoside coupling reagent. The coating is patternedinto features such that each feature has diameter of about 10 nm toabout 200 nm, with a center-to-center distance between two adjacentfeatures of about 10 nm to about 200 nm. In this instance, a pluralityof features accommodates a sessile drop volume of 0.2 pl during apolynucleotide synthesis deposition step. In some instances, a featurediameter of about 50 nm and a center-to-center distance between twoadjacent features of about 100 nm results in a feature density of about10 billion features per m² (1 feature/100×10⁻¹² m²).

A material deposition device described herein may comprises about 2,048nozzles that each deposit about 100,000 droplets per second at 1nucleobase per droplet. For each deposition device, at least about1.75×10¹³ nucleobases are deposited on the substrate per day. In someinstances, 100 to 500 nucleobase polynucleotides are synthesized. Insome instances, 200 nucleobase polynucleotides are synthesized.Optionally, over 3 days, at a rate of about 1.75×10¹³ bases per day, atleast about 262.5×10⁹ polynucleotides are synthesized.

In some arrangements, a device for application of one or more reagentsto a substrate during a synthesis reaction is configured to depositreagents and/or nucleotide monomers for nucleoside phosphoramidite basedsynthesis. Reagents for polynucleotide synthesis include reagents forpolynucleotide extension and wash buffers. As non-limiting examples, thedevice deposits cleaning reagents, coupling reagents, capping reagents,oxidizers, de-blocking agents, acetonitrile, phase change solvents,gases such as nitrogen gas, and any combination thereof. In addition,the device optionally deposits reagents for the preparation and/ormaintenance of substrate integrity. In some instances, thepolynucleotide synthesizer deposits a drop having a diameter less thanabout 200 μm, 100 μm, or 50 μm in a volume less than about 1000, 500,100, 50, or 20 pl. In some instances, the polynucleotide synthesizerdeposits between about 1 and 10,000, 1 and 5,000, 100 and 5,000, or1,000 and 5,000 droplets per second.

In some arrangement, during polynucleotide synthesis, the substrate ispositioned within and/or sealed within a flow cell. The flow cell mayprovide continuous or discontinuous flow of liquids such as thosecomprising reagents necessary for reactions within the substrate, forexample, oxidizers and/or solvents. The flow cell may provide continuousor discontinuous flow of a gas, such as nitrogen, for drying thesubstrate typically through enhanced evaporation of a volatilesubstrate. A variety of auxiliary devices are useful to improve dryingand reduce residual moisture on the surface of the substrate. Examplesof such auxiliary drying devices include, without limitation, a vacuumsource, depressurizing pump and a vacuum tank. In some instances, apolynucleotide synthesis system comprises one or more flow cells, suchas 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20 and one or more substrates, such as2, 3, 4, 5, 6, 7, 8, 9, 10 or 20. In some instances, a flow cell isconfigured to hold and provide reagents to the substrate during one ormore steps in a synthesis reaction. In some instances, a flowcellcomprises a lid that slides over the top of a substrate and can beclamped into place to form a pressure tight seal around the edge of thesubstrate. An adequate seal includes, without limitation, a seal thatallows for about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 atmospheres ofpressure. In some instances, the lid of the flow cell is opened to allowfor access to an application device such as a polynucleotidesynthesizer. In some instances, one or more steps of a polynucleotidesynthesis method are performed on a substrate within a flow cell,without the transport of the substrate.

In some arrangements, a device for treating a substrate with a fluidcomprises a spray bar. Nucleotide monomers may be applied onto asubstrate surface, and then a spray bar sprays the substrate surfacewith one or more treatment reagents using spray nozzles of the spraybar. In some arrangements, the spray nozzles are sequentially ordered tocorrelate with different treatment steps during polynucleotidesynthesis. The chemicals used in different process steps may be changedin the spray bar to readily accommodate changes in a synthesis method orbetween steps of a synthesis method. In some instances, the spray barcontinuously sprays a given chemistry on a surface of a substrate as thesubstrate moves past the spray bar. In some instances, the spray bardeposits over a wide area of a substrate, much like the spray bars usedin lawn sprinklers. In some instances, the spray bar nozzles arepositioned to provide a uniform coat of treatment material to a givenarea of a substrate.

In some instances, a polynucleotide synthesis system comprises one ormore elements useful for downstream processing of synthesizedpolynucleotides. As an example, the system comprises a temperaturecontrol element such as a thermal cycling device. In some instances, thetemperature control element is used with a plurality of resolvedreactors to perform nucleic acid assembly such as PCA and/or nucleicacid amplification such as PCR.

De Novo Polynucleotide Synthesis Using a Temperature Controllable DeviceProvided herein are methods for phase change applications to regulateaccess of reagents during polynucleotide synthesis in a temperaturespecific manner (see Table 1). Exemplary melting temperatures forsolvents described herein include about 10° C. to about 30° C., about10° C. to about 18° C., about −30° C. to about 40° C., or about 5° C. toabout 40° C. In some aspects, the phase change solvent has a meltingtemperature of about 15-16° C. In some aspects, the phase change solventhas a melting temperature of about 10-25° C. In some aspects, the phasechange solvent has a melting temperature of about 15-25° C. In someaspects, the phase change solvent has a melting temperature of about15-18° C. In some instances, the phase change solvent has a meltingtemperature of at least −20° C., or at least −15, −10, −5, 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or more than 40° C. In someinstances, the phase change solvent has a melting temperature no morethan −20° C., or no more than −15, −10, −5, 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, or no more than 40° C. In some instances, the phasechange solvent includes solvents containing about 5 to about 10 carbonatoms. In some instances, the phase change solvent is a polar solvent.In some instances, the phase change solvent is an ionic liquid. In someinstances, the phase change solvent is a supercritical fluid. In someinstances, the phase change solvent comprises one or more additives,such as salts, other solids, liquids, or dissolved gases that influencethe solvent properties. Exemplary densities for phase change solventsdescribed herein include about 0.5 g/mL to about 1.5 g/mL, or about 0.6g/mL to about 1.4 g/mL, or about 0.7 g/mL to about 1.3 g/mL. In someinstances, the phase change solvent is a solvent with a boiling point ofno more than 82° C. In some instances, the phase change solvent isacetonitrile or an acetonitrile mixture.

In some instances, the phase change solvent is trimethylacetonitrile(TMACN). Other exemplary phase change solvents include but are notlimited to trimethylacetonitrile (TMACN), dimethylsulfoxide (DMSO),p-xylene, cyclohexylcyanide, 2,5-dimethyl-2,4-hexadiene, cyclooctane,o-tolunitrile, acetophenone, cyclononane, p-methylbenzyl cyanide,propiophenone, m-nitrotoluene, o-dimethoxybenzene, m-chlorobenzaldehyde,o-chlorobenzaldehyde, cyclodecane, dimethyl succinate, butyrophenone,4-ethoxybenzaldehyde, m-tolyl acetate, phenyl propionate, or mixturesthereof.

TABLE 1 Phase Change Solvent Melting Temperature (° C.)trimethylacetonitrile (TMACN) 15 dimethylsulfoxide (DMSO) 18 p-xylene 13cyclohexylcyanide 11 2,5-dimethyl-2,4-hexadiene 13 cyclooctane 15o-tolunitrile 13 acetophenone 20 cyclononane 11 p-methylbenzyl cyanide18 propiophenone 19 m-nitrotoluene 16 o-dimethoxybenzene 15m-chlorobenzaldehyde 18 o-chlorobenzaldehyde 11 cyclodecane 10 dimethylsuccinate 19 butyrophenone 12 4-ethoxybenzaldehyde 14 m-tolyl acetate 12phenyl propionate 20

In some instances, a polynucleotide synthesis method used hereincomprises 1, 2, 3 or more sequential coupling steps. Prior to coupling,in many cases, the nucleoside bound to the substrate is de-protected byremoval of a protecting group, where the protecting group functions toprevent polymerization. A common protecting group is4,4′-dimethoxytrityl (DMT). In some instances, coupling steps arerepeated two or more times without removal of a protecting group.

Following coupling, phosphoramidite polynucleotide synthesis methodsoptionally comprise a capping step. In a capping step, the growingpolynucleotide is treated with a capping agent. A capping step generallyserves to block unreacted substrate-bound 5′-OH groups after couplingfrom further chain elongation, preventing the formation ofpolynucleotides with internal base deletions. Further, phosphoramiditesactivated with 1H-tetrazole often react, to a small extent, with the O6position of guanosine. Without being bound by theory, upon oxidationwith I₂/water, this side product, possibly via O6-N7 migration,undergoes depurination. The apurinic sites can end up being cleaved inthe course of the final deprotection of the polynucleotide thus reducingthe yield of the full-length product. The O6 modifications may beremoved by treatment with the capping reagent prior to oxidation withI₂/water. In some instances, inclusion of a capping step duringpolynucleotide synthesis decreases the error rate as compared tosynthesis without capping. As an example, the capping step comprisestreating the substrate-bound polynucleotide with a mixture of aceticanhydride and 1-methylimidazole. Following a capping step, the substrateis optionally washed.

Following addition of a nucleoside phosphoramidite, and optionally aftercapping and one or more wash steps, the substrate bound growing nucleicacid may be oxidized. The oxidation step comprises oxidizing thephosphite triester into a tetracoordinated phosphate triester, aprotected precursor of the naturally occurring phosphate diesterinternucleoside linkage. In some instances, oxidation of the growingpolynucleotide is achieved by treatment with iodine and water,optionally in the presence of a weak base such as a pyridine, lutidine,or collidine. Oxidation is sometimes carried out under anhydrousconditions using tert-Butyl hydroperoxide or(1S)-(+)-(10-camphorsulfonyl)-oxaziridine (CSO). In some methods, acapping step is performed following oxidation. A second capping stepallows for substrate drying, as residual water from oxidation that maypersist can inhibit subsequent coupling. Following oxidation, thesubstrate and growing polynucleotide is optionally washed. In someinstances, the step of oxidation is substituted with a sulfurizationstep to obtain polynucleotide phosphorothioates, wherein any cappingsteps can be performed after the sulfurization. Many reagents arecapable of the efficient sulfur transfer, including, but not limited to,3-(Dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3-thione, DDTT,3H-1,2-benzodithiol-3-one 1,1-dioxide, also known as Beaucage reagent,and N,N,N′N′-Tetraethylthiuram disulfide (TETD).

For a subsequent cycle of nucleoside incorporation to occur throughcoupling, a protected 5′ end of the substrate bound growingpolynucleotide must be removed so that the primary hydroxyl group canreact with a next nucleoside phosphoramidite. In some instances, theprotecting group is DMT and deblocking occurs with trichloroacetic acidin dichloromethane. Conducting detritylation for an extended time orwith stronger than recommended solutions of acids may lead to increaseddepurination of solid support-bound polynucleotide and thus reduces theyield of the desired full-length product. Methods and compositionsdescribed herein provide for controlled deblocking conditions limitingundesired depurination reactions. In some instances, the substrate boundpolynucleotide is washed after deblocking. In some instances, efficientwashing after deblocking contributes to synthesized polynucleotideshaving a low error rate.

Methods for the synthesis of polynucleotides on the substrates describedherein typically involve an iterating sequence of the following steps:application of a protected monomer to a surface of a substrate featureto link with either the surface, a linker or with a previouslydeprotected monomer; deprotection of the applied monomer so that it canreact with a subsequently applied protected monomer; and application ofanother protected monomer for linking. One or more intermediate stepsinclude oxidation and/or sulfurization. In some instances, one or morewash steps precede or follow one or all of the steps. In some instances,the last wash step comprises addition of a suitable phase changesolvent. In some instances, a coupling step occurs without the removalof a phase change solvent. In some instances the reaction solvent is aphase change solvent.

In some aspects of the methods described herein, the phase of thereaction solvent is used to block or unblock specific sites on thesurface of the device. In one example, the phase of the reaction solventat a site is controlled by one or more addressable heating elements, andmethods for the synthesis of polynucleotides comprises iteration of asequence of one or more of the following steps: deprotection of anapplied monomer or reactive group on the surface so that it can reactwith a subsequently applied protected monomer; optional cooling of thedevice surface; activation of all heater elements on the surface;addition of a phase change solvent, deactivation of all heating elementsat device sites to be blocked and application of a protected monomer toa surface of a substrate feature to link with either the surface, alinker or with a previously deprotected monomer. One or moreintermediate steps include activation of all heater elements, followedby oxidation and/or sulfurization. In some instances, one or more washsteps precede or follow one or all of the steps. In some instances,activation of all heating elements preceeds or follows one or more washsteps. In some instances, heating elements are activated at sites to beblocked.

Further described herein are methods wherein deactivation of one or moreheater elements at one or more surface regions for polynucleotidesynthesis causes solvent in these regions to freeze, forming a solid. Insome instances, the solid solvent solvent prevents contact of thesynthesis surface region with additional reagents such as detritylationreagents, preventing deprotection. In some instances, activation ofheating elements or deactivation of cooling elements causes the solid tomelt, allowing reagents to contact the synthesis surface.

Further described herein are methods wherein activation of one or moreheater elements at one or more surface regions for polynucleotidesynthesis causes solvent in these regions to boil, forming a gaseousbubble. In some instances, the bubble of gaseous solvent preventscontact of the synthesis surface region with additional reagents such asdetritylation reagents, preventing deprotection. In some instances,activation of cooling elements or deactivation of heating elementscauses the bubble to collapse, allowing reagents to contact thesynthesis surface.

In some instances, polynucleotides are synthesized with photolabileprotecting groups, where the hydroxyl groups generated on the surfaceare blocked by photolabile-protecting groups. When the surface isexposed to UV light, such as through a photolithographic mask, a patternof free hydroxyl groups on the surface may be generated. These hydroxylgroups can react with photoprotected nucleoside phosphoramidites,according to phosphoramidite chemistry. A second photolithographic maskcan be applied and the surface can be exposed to UV light to generatesecond pattern of hydroxyl groups, followed by coupling with5′-photoprotected nucleoside phosphoramidite. Likewise, patterns can begenerated and oligomer chains can be extended. Without being bound bytheory, the lability of a photocleavable group depends on the wavelengthand polarity of a solvent employed and the rate of photocleavage may beaffected by the duration of exposure and the intensity of light. Thismethod can leverage a number of factors such as accuracy in alignment ofthe masks, efficiency of removal of photo-protecting groups, and theyields of the phosphoramidite coupling step. Further, unintended leakageof light into neighboring sites can be minimized. The density ofsynthesized oligomer per spot can be monitored by adjusting loading ofthe leader nucleoside on the surface of synthesis.

The surface of the substrate that provides support for polynucleotidesynthesis may be chemically modified to allow for the synthesizedpolynucleotide chain to be cleaved from the surface. In some instances,the polynucleotide chain is cleaved at the same time as thepolynucleotide is deprotected. In some instances, the polynucleotidechain is cleaved after the polynucleotide is deprotected. In anexemplary scheme, a trialkoxysilyl amine such as (CH₃CH₂O)₃Si—(CH₂)₂—NH₂is reacted with surface SiOH groups of a substrate, followed by reactionwith succinic anhydride with the amine to create an amide linkage and afree OH on which the nucleic acid chain growth is supported. Cleavageincludes gas cleavage with ammonia or methylamine. In some instances,once released from the surface, polynucleotides are assembled intolarger nucleic acids that are sequenced and decoded to extract storedinformation.

Provided herein are systems and methods for synthesis of a high densityof polynucleotides on a substrate in a short amount of time. In someinstances, the substrate is a flexible substrate. In some instances, atleast about 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ bases are synthesizedin one day. In some instances, at least about 10×10⁸, 10×10⁹, 10×10¹⁰,10×10¹¹, or 10×10¹² polynucleotides are synthesized in one day. In someinstances, each polynucleotide synthesized comprises at least about 20,50, 100, 200, 300, 400 or 500 nucleobases. In some instances, thesebases are synthesized with a total average error rate of less than about1 in 100; 200; 300; 400; 500; 1000; 2000; 5000; 10000; 15000; 20000bases. In some instances, these error rates are for at least 50%, 60%,70%, 80%, 90%, 95%, 98%, 99%, 99.5%, or more of the polynucleotidessynthesized. In some instances, these at least 90%, 95%, 98%, 99%,99.5%, or more of the polynucleotides synthesized do not differ from apredetermined sequence for which they encode. In some instances, theerror rate for synthesized polynucleotides on a substrate using themethods and systems described herein is less than about 1 in 200. Insome instances, the error rate for synthesized polynucleotides on asubstrate using the methods and systems described herein is less thanabout 1 in 1,000. In some instances, the error rate for synthesizedpolynucleotides on a substrate using the methods and systems describedherein is less than about 1 in 2,000. In some instances, the error ratefor synthesized polynucleotides on a substrate using the methods andsystems described herein is less than about 1 in 3,000. In someinstances, the error rate for synthesized polynucleotides on a substrateusing the methods and systems described herein is less than about 1 in5,000. Individual types of error rates include mismatches, deletions,insertions, and/or substitutions for the polynucleotides synthesized onthe substrate. The term “error rate” refers to a comparison of thecollective amount of synthesized polynucleotide to an aggregate ofpredetermined polynucleotide sequences. In some instances, synthesizedpolynucleotides disclosed herein comprise a tether of 12 to 25 bases. Insome instances, the tether comprises 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more bases.

A suitable method for polynucleotide synthesis on a substrate of thisdisclosure is a phosphoramidite method comprising the controlledaddition of a phosphoramidite building block, i.e. nucleosidephosphoramidite, to a growing polynucleotide chain in a coupling stepthat forms a phosphite triester linkage between the phosphoramiditebuilding block and a nucleoside bound to the substrate (for example, anelongation step). In some instances, the nucleoside phosphoramidite isprovided to the substrate activated. In some instances, the nucleosidephosphoramidite is provided to the substrate with an activator. In someinstances, nucleoside phosphoramidites are provided to the substrate ina 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100-fold excess or more over thesubstrate-bound nucleosides. In some instances, the addition ofnucleoside phosphoramidite is performed in an anhydrous environment, forexample, in anhydrous acetonitrile. Following addition and linkage of anucleoside phosphoramidite in the coupling step, the substrate isoptionally washed. In some instances, the coupling step is repeated oneor more additional times, optionally with a wash step between nucleosidephosphoramidite additions to the substrate.

Nucleic Acid Assembly

Polynucleotides may be designed to collectively span a large region of apredetermined sequence that encodes for information. In some instances,larger polynucleotides are generated through ligation reactions to jointhe synthesized polynucleotides. One example of a ligation reaction ispolymerase chain assembly (PCA). In some instances, at least of aportion of the polynucleotides are designed to include an appendedregion that is a substrate for universal primer binding. For PCAreactions, the presynthesized polynucleotides include overlaps with eachother (e.g., 4, 20, 40 or more bases with overlapping sequence). Duringthe polymerase cycles, the polynucleotides anneal to complementaryfragments and then are filled in by polymerase. Each cycle thusincreases the length of various fragments randomly depending on whichpolynucleotides find each other. Complementarity amongst the fragmentsallows for forming a complete large span of double-stranded DNA. In someinstances, after the PCA reaction is complete, an error correction stepis conducted using mismatch repair detecting enzymes to removemismatches in the sequence. Once larger fragments of a target sequenceare generated, they can be amplified. For example, in some instances, atarget sequence comprising 5′ and 3′ terminal adapter sequences isamplified in a polymerase chain reaction (PCR) which includes modifiedprimers that hybridize to the adapter sequences. In some instances, themodified primers comprise one or more uracil bases. The use of modifiedprimers allows for removal of the primers through enzymatic reactionscentered on targeting the modified base and/or gaps left by enzymeswhich cleave the modified base pair from the fragment. What remains is adouble-stranded amplification product that lacks remnants of adaptersequence. In this way, multiple amplification products can be generatedin parallel with the same set of primers to generate different fragmentsof double-stranded DNA.

Error correction may be performed on synthesized polynucleotides and/orassembled products. An example strategy for error correction involvessite-directed mutagenesis by overlap extension PCR to correct errors,which is optionally coupled with two or more rounds of cloning andsequencing. In certain instances, double-stranded nucleic acids withmismatches, bulges and small loops, chemically altered bases and/orother heteroduplexes are selectively removed from populations ofcorrectly synthesized nucleic acids. In some instances, error correctionis performed using proteins/enzymes that recognize and bind to or nextto mismatched or unpaired bases within double-stranded nucleic acids tocreate a single or double-strand break or to initiate a strand transfertransposition event. Non-limiting examples of proteins/enzymes for errorcorrection include endonucleases (T7 Endonuclease I, E. coliEndonuclease V, T4 Endonuclease VII, mung bean nuclease, Cell, E. coliEndonuclease IV, UVDE), restriction enzymes, glycosylases,ribonucleases, mismatch repair enzymes, resolvases, helicases, ligases,antibodies specific for mismatches, and their variants. Examples ofspecific error correction enzymes include T4 endonuclease 7, T7endonuclease 1, S1, mung bean endonuclease, MutY, MutS, MutH, MutL,cleavase, CELI, and HINF1. In some instances, DNA mismatch-bindingprotein MutS (Thermus aquaticus) is used to remove failure products froma population of synthesized products. In some instances, errorcorrection is performed using the enzyme Correctase. In some instances,error correction is performed using SURVEYOR endonuclease(Transgenomic), a mismatch-specific DNA endonuclease that scans forknown and unknown mutations and polymorphisms for heteroduplex DNA.

Computer Systems

In various aspects, any of the systems described herein are operablylinked to a computer and are optionally automated through a computereither locally or remotely. In various instances, the methods andsystems of the invention further comprise software programs on computersystems and use thereof. Accordingly, computerized control for thesynchronization of the dispense/vacuum/refill functions such asorchestrating and synchronizing the material deposition device movement,dispense action and vacuum actuation are within the bounds of theinvention. In some instances, the computer systems are programmed tointerface between the user specified base sequence and the position of amaterial deposition device to deliver the correct reagents to specifiedregions of the substrate.

The computer system 900 illustrated in FIG. 9 may be understood as alogical apparatus that can read instructions from media 911 and/or anetwork port 905, which can optionally be connected to server 909. Thesystem, such as shown in FIG. 9 can include a CPU 901, disk drives 903,optional input devices such as keyboard 915 and/or mouse 916 andoptional monitor 907. Data communication can be achieved through theindicated communication medium to a server at a local or a remotelocation. The communication medium can include any means of transmittingand/or receiving data. For example, the communication medium can be anetwork connection, a wireless connection or an internet connection.Such a connection can provide for communication over the World Wide Web.It is envisioned that data relating to the present disclosure can betransmitted over such networks or connections for reception and/orreview by a party 922.

FIG. 10 is a block diagram illustrating a first example architecture ofa computer system 1000 that can be used in connection with exampleinstances of the present invention. As depicted in FIG. 10, the examplecomputer system can include a processor 1002 for processinginstructions. Non-limiting examples of processors include: Intel Xeon™processor, AMD Opteron™ processor, Samsung 32-bit RISC ARM 1176JZ(F)-Sv1.0™ processor, ARM Cortex-A8 Samsung S5PC100™ processor, ARM Cortex-A8Apple A4™ processor, Marvell PXA 930™ processor, or afunctionally-equivalent processor. Multiple threads of execution can beused for parallel processing. In some instances, multiple processors orprocessors with multiple cores can also be used, whether in a singlecomputer system, in a cluster, or distributed across systems over anetwork comprising a plurality of computers, cell phones, and/orpersonal data assistant devices.

As illustrated in FIG. 10, a high speed cache 1004 can be connected to,or incorporated in, the processor 1002 to provide a high speed memoryfor instructions or data that have been recently, or are frequently,used by processor 1002. The processor 1002 is connected to a northbridge 1006 by a processor bus 1008. The north bridge 1006 is connectedto random access memory (RAM) 1010 by a memory bus 1012 and managesaccess to the RAM 1010 by the processor 1002. The north bridge 1006 isalso connected to a south bridge 1014 by a chipset bus 1016. The southbridge 1014 is, in turn, connected to a peripheral bus 1018. Theperipheral bus can be, for example, PCI, PCI-X, PCI Express, or otherperipheral bus. The north bridge and south bridge are often referred toas a processor chipset and manage data transfer between the processor,RAM, and peripheral components on the peripheral bus 1018. In somealternative architectures, the functionality of the north bridge can beincorporated into the processor instead of using a separate north bridgechip.

In some instances, system 1000 can include an accelerator card 1022attached to the peripheral bus 1018. The accelerator can include fieldprogrammable gate arrays (FPGAs) or other hardware for acceleratingcertain processing. For example, an accelerator can be used for adaptivedata restructuring or to evaluate algebraic expressions used in extendedset processing.

Software and data are stored in external storage 1024 and can be loadedinto RAM 1010 and/or cache 1004 for use by the processor. The system1000 includes an operating system for managing system resources;non-limiting examples of operating systems include: Linux, Windows™,MACOS™, BlackBerry OS™, iOS™, and other functionally-equivalentoperating systems, as well as application software running on top of theoperating system for managing data storage and optimization inaccordance with example instances of the present invention.

In this example, system 1000 also includes network interface cards(NICs) 1020 and 1021 connected to the peripheral bus for providingnetwork interfaces to external storage, such as Network Attached Storage(NAS) and other computer systems that can be used for distributedparallel processing.

FIG. 11 is a diagram showing a network 1100 with a plurality of computersystems 1102 a, and 1102 b, a plurality of cell phones and personal dataassistants 1102 c, and Network Attached Storage (NAS) 1104 a, and 1104b. In example instances, systems 1102 a, 1102 b, and 1102 c can managedata storage and optimize data access for data stored in NetworkAttached Storage (NAS) 1104 a and 1104 b. A mathematical model can beused for the data and be evaluated using distributed parallel processingacross computer systems 1102 a, and 1102 b, and cell phone and personaldata assistant systems 1102 c. Computer systems 1102 a, and 1102 b, andcell phone and personal data assistant systems 1102 c can also provideparallel processing for adaptive data restructuring of the data storedin Network Attached Storage (NAS) 1104 a and 1104 b. FIG. 11 illustratesan example only, and a wide variety of other computer architectures andsystems can be used in conjunction with the various instances of thepresent invention. For example, a blade server can be used to provideparallel processing. Processor blades can be connected through a backplane to provide parallel processing. Storage can also be connected tothe back plane or as Network Attached Storage (NAS) through a separatenetwork interface.

In some example instances, processors can maintain separate memoryspaces and transmit data through network interfaces, back plane or otherconnectors for parallel processing by other processors. In otherinstances, some or all of the processors can use a shared virtualaddress memory space.

FIG. 12 is a block diagram of a multiprocessor computer system 1200using a shared virtual address memory space in accordance with anexample embodiment. The system includes a plurality of processors 1202a-f that can access a shared memory subsystem 1204. The systemincorporates a plurality of programmable hardware memory algorithmprocessors (MAPs) 1206 a-f in the memory subsystem 1204. Each MAP 1206a-f can comprise a memory 1208 a-f and one or more field programmablegate arrays (FPGAs) 1210 a-f The MAP provides a configurable functionalunit and particular algorithms or portions of algorithms can be providedto the FPGAs 1210 a-f for processing in close coordination with arespective processor. For example, the MAPs can be used to evaluatealgebraic expressions regarding the data model and to perform adaptivedata restructuring in example instances. In this example, each MAP isglobally accessible by all of the processors for these purposes. In oneconfiguration, each MAP can use Direct Memory Access (DMA) to access anassociated memory 1208 a-f, allowing it to execute tasks independentlyof, and asynchronously from, the respective microprocessor 1202 a-f Inthis configuration, a MAP can feed results directly to another MAP forpipelining and parallel execution of algorithms.

The above computer architectures and systems are examples only, and awide variety of other computer, cell phone, and personal data assistantarchitectures and systems can be used in connection with exampleinstances, including systems using any combination of generalprocessors, co-processors, FPGAs and other programmable logic devices,system on chips (SOCs), application specific integrated circuits(ASICs), and other processing and logic elements. In some instances, allor part of the computer system can be implemented in software orhardware. Any variety of data storage media can be used in connectionwith example instances, including random access memory, hard drives,flash memory, tape drives, disk arrays, Network Attached Storage (NAS)and other local or distributed data storage devices and systems.

The following examples are set forth to illustrate more clearly theprinciple and practice of instances disclosed herein to those skilled inthe art and are not to be construed as limiting the scope of any claimedinstances. Unless otherwise stated, all parts and percentages are on aweight basis.

EXAMPLES Example 1: Synthesis of 50-Mer Sequence Polynucleotides on aTemperature Controllable Surface Utilizing a Phase Change Solvent

A polynucleotide synthesis device 101 (see FIG. 1), is assembled into atemperature controllable flowcell (FIG. 3A and FIG. 3B), which isconnected to a flowcell (Applied Biosystems (ABI394 DNA Synthesizer”) asshown in in FIG. 7. The bottom surface of the well 305 (FIG. 3A and FIG.3B), coated with SiO₂, is uniformly functionalized withN-(3-TRIETHOXYSILYLPROPYL)-4-HYDROXYBUTYRAMIDE (Gelest, CAS No.156214-80-1) and is used to synthesize an exemplary polynucleotide of 50bp (“50-mer polynucleotide”) using polynucleotide synthesis methodsdescribed herein. The sequence of the 50-mer is as described in SEQ IDNO.: 1. 5′AGACAATCAACCATTTGGGGTGGACAGCCTTGACCTCTAGACTTCGGCAT##TTTTTTTTTT3′ (SEQ ID NO.: 1), where # denotes Thymidine-succinyl hexamide CEDphosphoramidite (CLP-2244 from ChemGenes), which is a cleavable linkerenabling the release of polynucleotides from the surface duringdeprotection.

The synthesis is done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 2and an ABI synthesizer, with modification: the chemical couplingreaction in each cell is controlled via a temperature controllablesurface (see FIG. 8). The device temperature is lowered to about 5° C.below the freezing temperature of a phase change solvent with a coldchuck. During the coupling step, the phase change solvent is addedimmediately prior to addition of coupling reagents, and the phase changesolvent freezes. Wells are activated for coupling by heating throughaddressable heating elements, which melt the solvent in individualwells. Remaining wells with frozen solvent do not react with liquidcoupling reagents, and are inactive. Coupling steps are iterated, eachwith different DNA bases, for example A, T, G, and C, while changing theactive wells for each coupling iteration. After all desired sites havebeen functionalized, all wells are activated by heating; global capping,oxidation, and deblocking are then conducted. This overall process isrepeated until polynucleotides of the desired length are synthesized.

TABLE 2 Step Description 0 Deblock 1 Flush surface 2 Activate all heaterelements 3 Flow phase change reagent 4 Deactivate heaters at devicesites to be blocked 5 Flow nucleotide A and activator 6 Flush 7 Activateall heater elements 8 Flush 9 Flow phase change reagent 10 Deactivateheaters at device sites to be blocked 11 Flow nucleotide G and activator12 Flush 13 Activate all heater elements 14 Flush 15 Flow phase changereagent 16 Deactivate heaters at device sites to be blocked 17 Flownucleotide C and activator 18 Flush 19 Activate all heater elements 20Flush 21 Flow phase change reagent 22 Deactivate heaters at device sitesto be blocked 23 Flow nucleotide T and activator 24 Flush 25 Activateall heater elements 26 Flush 27 Oxidation

The device temperature is lowered to about 5° C. below the freezingtemperature of a phase change solvent with a cold chuck. During thecoupling step, the phase change solvent is added immediately prior toaddition of coupling reagents, and the phase change solvent freezes.Wells are activated for coupling by heating through addressable heatingelements, which melt the solvent in individual wells. Remaining wellswith frozen solvent do not react with liquid coupling reagents, and areinactive towards coupling reagents. Coupling steps are iterated withdifferent DNA bases, for example A, T, G, and C, while changing theactive wells for each coupling iteration. After all desired sites havebeen functionalized, all wells are activated by heating; global capping,oxidation, and deblocking are then conducted. This overall process isrepeated until polynucleotides of the desired length are synthesized.

Example 2: Synthesis of a 50-Mer Sequence Polynucleotides on aTemperature Controllable Surface Utilizing a Solvent Vapor Bubble

A polynucleotide synthesis device 101 (see FIG. 1), is assembled into atemperature controllable flowcell (FIG. 4A), which is connected to aflowcell (Applied Biosystems (ABI394 DNA Synthesizer”) as shown in inFIG. 7. The bottom surface of the well 401 (FIG. 4A), is functionalizedusing the general methods from Example 1.

The synthesis is done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer, with modification: the chemical couplingreaction in each cell is controlled via a temperature controllablesurface (see FIG. 8).

TABLE 3 Step Description 0 Deblock 1 Flush surface 2 Deactivate allheater elements 3 Activate heaters at device sites to be blocked 4 Flownucleotide A and activator 5 Flush 6 Deactivate all heater elements 7Flush 8 Activate heaters at device sites to be blocked 9 Flow nucleotideG and activator 10 Flush 11 Deactivate all heater elements 12 Flush 13Activate heaters at device sites to be blocked 14 Flow nucleotide C andactivator 15 Flush 16 Deactivate all heater elements 17 Flush 18Activate heaters at device sites to be blocked 19 Flow nucleotide T andactivator 20 Flush 21 Deactivate all heater elements 22 Flush 23Oxidation

Wells are blocked against coupling by heating through addressableheating elements, which vaporizes the solvent in individual wells,creating a bubble 417. Remaining wells with liquid solvent contactingthe polynucleotide surface 407 for extension or synthesis react withliquid coupling reagents, and are active. Coupling steps are iteratedwith different DNA bases, for example A, T, G, and C, while changing theactive wells for each coupling iteration. Inactive wells are activatedby turning off heating elements at those wells, causing the vaporbubbles to collapse. After all desired sites have been functionalized,all wells are activated by turning off heating elements; global capping,oxidation, and deblocking are then conducted. This overall process isrepeated until polynucleotides of the desired length are synthesized.

Example 3: Synthesis of 50-Mer Sequence Polynucleotides on a TemperatureControllable Surface Utilizing Nanoposts

A polynucleotide synthesis device 101 (see FIG. 1), is assembled into atemperature controllable flowcell (FIG. 4B), which is connected to aflowcell (Applied Biosystems (ABI394 DNA Synthesizer”) as shown in inFIG. 7. The top surface of the nanopost 407 (FIG. 4B), is functionalizedusing the general methods from Example 1.

The synthesis is done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer, with modification: the chemical couplingreaction in each cell is controlled via a temperature controllablesurface (see FIG. 8), using the general method of Example 2.

Example 4: Synthesis of 50-Mer Sequence Polynucleotides on a TemperatureControllable Surface Utilizing Nanowires

A polynucleotide synthesis device 101 (see FIG. 1), is assembled into atemperature controllable flowcell (FIG. 5), which is connected to aflowcell (Applied Biosystems (ABI394 DNA Synthesizer”) as shown in inFIG. 7. The surface of the nanorods 502 (FIG. 5), is functionalizedusing the general methods from Example 1.

The synthesis is done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 3and an ABI synthesizer, with modification: the chemical couplingreaction in each cell is controlled via a temperature controllablesurface (see FIG. 8), using the general methods of Example 2.

Example 5: Synthesis of 50-Mer Sequence Polynucleotides on a SurfaceContaining Nanorods and Utilizing a Phase Change Solvent

A polynucleotide synthesis device 101 (see FIG. 1), is assembled into atemperature controllable flowcell (FIG. 6), which is connected to aflowcell (Applied Biosystems (ABI394 DNA Synthesizer”) as shown in inFIG. 7. The surface of the nanorods 603 (FIG. 6), is functionalizedusing the general methods from Example 1.

The synthesis is done using standard DNA synthesis chemistry (coupling,capping, oxidation, and deblocking) according to the protocol in Table 4and an ABI synthesizer, with modification: the chemical couplingreaction in each cell is controlled via a temperature controllablesurface (see FIG. 8). During the coupling step, the phase change solventis added immediately prior to addition of coupling reagents. Wells aredeactivated for coupling by cooling through an addressable lower contact601 connected to one or more nanorods, which freezes a layer of solventaround the nanorods. Remaining nanorods with liquid solvent react withliquid coupling reagents, and are active. Coupling steps are iteratedwith different DNA bases, for example A, T, G, and C, while changing theactive wells for each coupling iteration. After all desired sites havebeen functionalized, all nanorods are activated by discontinuingcooling; global capping, oxidation, and deblocking are then conducted.This overall process is repeated until polynucleotides of the desiredlength are synthesized.

TABLE 4 Step Description 0 Deblock 1 Flush surface 2 Deactivate allcooling elements 3 Flow phase change reagent 4 Activate cooling atdevice sites to be blocked 5 Flow nucleotide A and activator 6 Flush 7Deactivate all cooling elements 8 Flush 9 Flow phase change reagent 10Activate cooling at device sites to be blocked 11 Flow nucleotide G andactivator 12 Flush 13 Deactivate all cooling elements 14 Flush 15 Flowphase change reagent 16 Activate cooling at device sites to be blocked17 Flow nucleotide C and activator 18 Flush 19 Deactivate all coolingelements 20 Flush 21 Flow phase change reagent 22 Activate cooling atdevice sites to be blocked 23 Flow nucleotide T and activator 24 Flush25 Deactivate all cooling elements 26 Flush 27 Oxidation

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

1.-41. (canceled)
 42. A method for polynucleotide synthesis, the methodcomprising: a. providing predetermined sequences for a library ofpolynucleotides; b. providing a substrate comprising a surface, whereinthe surface comprises a solvent; c. synthesizing the library ofpolynucleotides extending from the surface, wherein the solvent when ina gas phase prevents deblocking of at least one polynucleotide extendingfrom the at least one region of the surface.
 43. The method of claim 42,wherein synthesizing the library of polynucleotides extending from thesurface comprises contacting the surface with a first nucleosidephosphoramidite.
 44. The method of claim 43, wherein synthesizing thelibrary of polynucleotides extending from the surface further comprisescontacting the surface with a second nucleoside phosphoramidite, whereinthe solvent is not removed between contact with the first nucleotidephosphoramidite and the second nucleotide phosphoramidite.
 45. Themethod of claim 42, wherein synthesizing the library of polynucleotidesextending from the surface further comprises condensing the solventpresent at the at least one region of the surface, and deblocking atleast one extended polynucleotide extending from the surface in the atleast one region.
 46. The method of claim 42, wherein the solvent has aboiling temperature no more than about 75 degrees C.
 47. The method ofclaim 46, wherein the solvent has a boiling temperature no more thanabout 65 degrees C.
 48. The method of claim 42, wherein the solvent hasa boiling temperature of between 0 degrees C. to 82 degrees C.
 49. Themethod of claim 48, wherein the solvent has a boiling temperature ofbetween 30 degrees C. to 82 degrees C.
 50. The method of claim 49,wherein the solvent has a boiling temperature of between 55 degrees C.to 82 degrees C.
 51. The method of claim 42, wherein the surfacecomprises at least 30,000 loci for nucleic acid synthesis.
 52. Themethod of claim 51, wherein the surface comprises at least 50,000 locifor nucleic acid synthesis.
 53. The method of claim 52, wherein thesurface comprises at least 100,000 loci for nucleic acid synthesis. 54.The method of claim 53, wherein the surface comprises at least 200,000loci for nucleic acid synthesis.
 55. The method of claim 54, wherein thesurface comprises at least 1,000,000 loci for nucleic acid synthesis.56. The method of claim 42, wherein the solvent has a boilingtemperature no more than about 82 degrees C.
 57. The method of claim 42,wherein the solvent is a polar solvent.
 58. The method of claim 57,wherein the solvent is acetonitrile.
 59. The method of claim 42, whereinthe solvent is a non-polar solvent.
 60. The method of claim 42, whereinthe solvent when in a liquid phase allows deblocking of at least onepolynucleotide extending from the at least one region of the surface.61. The method of claim 42, wherein the method further comprisescondensing the solvent after deblocking of at least one polynucleotideextending from the at least one region of the surface.